Compositions and methods for treatment of diseases and disorders associated with cytokine signaling

ABSTRACT

Compositions and methods are provided for the diagnosis and treatment of inflammation and autoimmune disorders, such as psoriasis. Compositions and methods for modulating IL-23 or IL-22 signaling are provided.

This application claims the benefit of U.S. Provisional Application No.60/741,640, filed Dec. 2, 2005, and U.S. Provisional Application No.60/822,597, filed Aug. 16, 2006, the disclosures of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful for thediagnosis and treatment of diseases and disorders associated withcytokine signaling.

BACKGROUND OF THE INVENTION

Various diseases and disorders are associated with inflammation.Inflammation is a process associated with recruitment of inflammatorycells (e.g., leukocytes) to a site of injury or infection. Inflammationgenerally protects the body from infection and injury. However,excessive or inappropriate inflammation can have deleterious effects.Autoimmune disorders, for example, often trigger inflammation resultingin the destruction of normal body tissues. Inflammation is also linkedto cancer. See, e.g., Coussens et al. (2002) Nature 420:860-867. Forexample, chronic inflammation associated with inflammatory bowel disease(IBD) is strongly correlated with colon carcinogenesis. During theinflammatory response, certain inflammatory cells produce agents thatpromote angiogenesis, reduce the anti-tumor activity of cytotoxicT-cells, and induce mutations in DNA, thus creating an environment thepromotes tumor progression. Id.

IL-23 is a heterodimeric cytokine that plays a dominant role inautoimmune/inflammatory disorders, and in particular, chronicinflammation. For example, studies in mice have revealed that IL-23 isessential for development of experimental allergic encephalomyelitis(autoimmune inflammation of the brain), which is a model for multiplesclerosis; collagen-induced arthritis, which is a model for rheumatoidarthritis; and delayed-type hypersensitivity. IL-23 also functions tomaintain established colitis (a form of IBD). Transgenic expression ofIL-23 leads to systemic inflammatory response, and dysregulation ofIL-23 leads to eczematous skin disease (an inflammatory skin condition).IL-23 stimulates a unique population of T cells (Th_(IL-17) cells),which in turn induce the production of IL-17 and proinflammatorycytokines. For review of the roles of IL-23 in inflammation andautoimmunity, see, e.g., Hunter (2005) Nat. Rev. Immunol. 5:521-531; andHolscher (2005) Curr. Opin. Invest. Drugs 6:489-495. IL-23 has also beenshown to promote tumor growth by increasing angiogenesis and decreasingtumor infiltration by cytotoxic T cells. Langowski et al. (2006) Nature442:461-465.

SUMMARY OF THE INVENTION

Compositions and methods useful for the diagnosis and treatment ofinflammatory disorders and autoimmune disorders (e.g., psoriasis) areprovided. Compositions and methods useful for the modulating IL-23 orIL-22 signaling are further provided. These and other embodiments of theinvention are provided herein. The present invention is based, in part,on the elucidation of a signaling pathway in which IL-23 acts throughIL-22 by inducing IL-22 expression from a recently discovered subset ofhelper T cells (Th cells), i.e., the Th_(IL-17) lineage.

In one aspect, an antibody that specifically binds to IL-22 is provided,wherein the antibody is (a) an antibody produced by a hybridoma selectedfrom 3F11.3 (ATCC Accession No. PTA-7312), hybridoma 11H4.4 (ATCCAccession No. PTA-7315), and hybridoma 8E11.9 (ATCC Accession No.PTA-7319); (b) an affinity matured form of the antibody of (a); (c) anantigen-binding fragment of the antibody of (a) or (b); or (d) ahumanized form of the antibody of (a), (b), or (c).

In another aspect, an antibody that specifically binds to IL-22R isprovided, wherein the antibody is (a) an antibody produced by ahybridoma selected from 7E9 (ATCC Accession No. PTA-7313), hybridoma8A12 (ATCC Accession No. PTA-7318), and hybridoma 8H11 (ATCC AccessionNo. PTA-7317); (b) an affinity matured form of the antibody of (a); (c)an antigen-binding fragment of the antibody of (a) or (b); or (d) ahumanized form of the antibody of (a), (b), or (c).

In another aspect, a method of treating an autoimmune disorder isprovided, wherein the autoimmune disorder is not arthritis, the methodcomprising administering to a mammal an effective amount of apharmaceutical formulation comprising an antagonist of IL-22. In onesuch embodiment, the IL-22 antagonist is an antibody that specificallybinds IL-22. In one embodiment, the antibody that specifically bindsIL-22 is (a) an antibody produced by a hybridoma selected from 3F11.3(ATCC Accession No. PTA-7312), hybridoma 11H4.4 (ATCC Accession No.PTA-7315), and hybridoma 8E11.9 (ATCC Accession No. PTA-7319); (b) anaffinity matured form of the antibody of (a); (c) an antigen-bindingfragment of the antibody of (a) or (b); or (d) a humanized form of theantibody of (a), (b), or (c). In one embodiment, the IL-22 antagonist isan antibody that specifically binds IL-22R. In one such embodiment, theantibody that specifically binds IL-22R is (a) an antibody produced by ahybridoma selected from 7E9 (ATCC Accession No. PTA-7313), hybridoma8A12 (ATCC Accession No. PTA-7318), and hybridoma 8H11 (ATCC AccessionNo. PTA-7317); (b) an affinity matured form of the antibody of (a); (c)an antigen-binding fragment of the antibody of (a) or (b); or (d) ahumanized form of the antibody of (a), (b), or (c). In one embodiment,the IL-22 antagonist is IL-22BP. In one embodiment, the autoimmunedisorder is inflammatory bowel disease. In one embodiment, theautoimmune disorder is psoriasis. In one embodiment, the method furthercomprises administering at least one antibody selected from an antibodythat specifically binds IL20Ra, an antibody that specifically bindsIL20Rb, and an antibody that specifically binds IL-22R. In oneembodiment, the method further comprises administering at least oneantibody selected from an antibody that specifically binds IL-22, anantibody that specifically binds IL20Ra, and an antibody thatspecifically binds IL20Rb.

In another aspect, a method of treating inflammation is provided,wherein the inflammation is not arthritic inflammation, the methodcomprising administering to a mammal an effective amount of apharmaceutical formulation comprising an antagonist of IL-22. In oneembodiment, the IL-22 antagonist is an antibody that specifically bindsIL-22. In one such embodiment, the antibody that specifically bindsIL-22 is (a) an antibody produced by a hybridoma selected from 3F11.3(ATCC Accession No. PTA-7312), hybridoma 11H4.4 (ATCC Accession No.PTA-7315), and hybridoma 8E11.9 (ATCC Accession No. PTA-7319); (b) anaffinity matured form of the antibody of (a); (c) an antigen-bindingfragment of the antibody of (a) or (b); or (d) a humanized form of theantibody of (a), (b), or (c). In one embodiment, the IL-22 antagonist isan antibody that specifically binds IL-22R. In one such embodiment, theantibody that specifically binds IL-22R is (a) an antibody produced by ahybridoma selected from 7E9 (ATCC Accession No. PTA-7313), hybridoma8A12 (ATCC Accession No. PTA-7318), and hybridoma 8H11 (ATCC AccessionNo. PTA-7317); (b) an affinity matured form of the antibody of (a); (c)an antigen-binding fragment of the antibody of (a) or (b); or (d) ahumanized form of the antibody of (a), (b), or (c). In one embodiment,the IL-22 antagonist is IL-22BP. In one embodiment, the inflammation isautoimmune inflammation. In one embodiment, the inflammation is skininflammation. In one embodiment, the inflammation is chronicinflammation.

In another aspect, a method of inhibiting tumor progression is provided,the method comprising administering to a mammal an effective amount of apharmaceutical formulation comprising an antagonist of IL-22. In oneembodiment, the IL-22 antagonist is an antibody that specifically bindsIL-22. In one such embodiment, the antibody that specifically bindsIL-22 is (a) an antibody produced by a hybridoma selected from 3F11.3(ATCC Accession No. PTA-7312), hybridoma 11H4.4 (ATCC Accession No.PTA-7315), and hybridoma 8E11.9 (ATCC Accession No. PTA-7319); (b) anaffinity matured form of the antibody of (a); (c) an antigen-bindingfragment of the antibody of (a) or (b); or (d) a humanized form of theantibody of (a), (b), or (c). In one embodiment, the IL-22 antagonist isan antibody that specifically binds IL-22R. In one such embodiment, theantibody that specifically binds IL-22R is (a) an antibody produced by ahybridoma selected from 7E9 (ATCC Accession No. PTA-7313), hybridoma8A12 (ATCC Accession No. PTA-7318), and hybridoma 8H11 (ATCC AccessionNo. PTA-7317); (b) an affinity matured form of the antibody of (a); (c)an antigen-binding fragment of the antibody of (a) or (b); or (d) ahumanized form of the antibody of (a), (b), or (c). In one embodiment,the IL-22 antagonist is IL-22BP.

In another aspect, a method of stimulating an IL-23-mediated signalingpathway in a biological system is provided, the method comprisingproviding an IL-22 agonist to the biological system. In one embodiment,the IL-22 agonist is IL-22. In another aspect, a method of inhibiting anIL-23-mediated signaling pathway in a biological system is provided, themethod comprising providing an IL-22 antagonist to the biologicalsystem. In one embodiment, the IL-22 antagonist is an antibody thatspecifically binds IL-22. In one embodiment, the IL-22 antagonist is anantibody that specifically binds IL-22R.

In another aspect, a method of stimulating a Th_(IL-17) cell function isprovided, the method comprising exposing a Th_(IL-17) cell to an IL-22agonist. In one embodiment, the IL-22 agonist is IL-22. In anotheraspect, a method of inhibiting a cell function is provided, the methodcomprising exposing a Th_(IL-17) cell to an IL-22 antagonist. In oneembodiment, the IL-22 antagonist is an antibody that specifically bindsIL-22. In one embodiment, the IL-22 antagonist is an antibody thatspecifically binds IL-22R.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a cDNA encoding anative human IL-22.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows an amino acid sequence (SEQ ID NO:3) of a native humanIL-22R.

FIG. 4 shows an amino acid sequence (SEQ ID NO:4) of a native humanIL-22BP.

FIG. 5 is a list of all IL-22 antibodies generated and their respectiveproperties, as described in Example 1. Intracellular staining isabbreviated as IC.

FIG. 6 shows that anti-IL-22 antibodies are able to block STAT3activation, as described in Example 2.

FIG. 7 shows that three specific anti-IL-22 antibodies block human IL-22in a dose dependent manner, as described in Example 3.

FIG. 8 shows that three specific anti-IL-22 antibodies are able to blockmurine IL-22 in a dose dependent manner, as described in Example 4.

FIG. 9 is a calculation of the affinity of anti-IL-22 antibodies forhuman IL-22, as described in Example 5.

FIG. 10 shows that anti-IL-22 antibodies detect intracellular expressionof IL-22, as described in Example 6.

FIG. 11 shows intracellular FACS staining for IL-22 using labeledanti-IL-22 antibodies, as described in Example 6.

FIG. 12 shows expression of IL-22 in murine Th1 cells as determined by5′ nuclease analysis, as described in Example 7.

FIG. 13 shows expression of IL-22 in murine γδ T cells as determined by5′ nuclease analysis, as described in Example 8.

FIG. 14 shows expression of IL-22 in activated human T cells asdetermined by microarray analysis, as described in Example 9.

FIG. 15 shows the expression level of IL-22 in T cells by FACS, asdescribed in Example 10.

FIG. 16 shows the testing of anti-IL-22R antibodies on 293 cellsexpressing IL-22R, as described in Example 11.

FIG. 17 shows that anti-IL-22R antibodies can block IL-22-inducedexpression of a STAT3 reporter construct, as described in Example 12.

FIG. 18 shows expression of IL-22R and IL-10R2 on the surface of primarykeratinocytes, as described in Example 13.

FIG. 19 shows that IL-22 induces thicking of human epidermis, asdescribed in Example 14.

FIG. 20 shows that IL-22 induces cytokeratin 16 expression, a marker forkeratinocyte turnover, as described in Example 14.

FIG. 21 shows that treatment of human epidermis with IL-22 causesinduction of psoriasin expression, a gene highly expressed in psoriasis,as described in Example 14.

FIG. 22 shows that treatment of keratinocytes with IL-22 elevates theexpression of several genes, including psoriasin, as described inExample 15.

FIG. 23 shows that psoriasin expression is reduced by treatment withanti-IL-22 and anti-IL-22R antibodies, as described in Example 14.

FIG. 24 shows that epidermal thickening is reduced by treatment withanti-IL-22 and anti-IL-22R antibodies, as described in Example 14.

FIG. 25 shows that epidermal thickening is reduced by treatment withanti-IL-22 and anti-IL-22R antibodies, as described in Example 14.

FIG. 26 shows that IL-23 and IL-12 induce epidermal thickening withdistinct histological features, as described in Example 16.

FIG. 27 shows that IL-23 induces expression of IL-22, and IL-22 inducesdermal inflammation and epidermal thickening in vivo, as described inExamples 17 and 18.

FIG. 28 shows that IL-12 and IL-23 induce expression of distinct sets ofcytokines, as described in Example 17.

FIG. 29 shows that treatment with an anti-IL-22 monoclonal antibodysignificantly reduces IL-23-induced epidermal acanthosis in vivo, asdescribed in Example 20.

FIG. 30 shows the strategy used to disrupt the IL-22 gene in mice andevidence confirming that IL-22 expression is absent in IL-22^(−/−) mice,as described in Example 20.

FIG. 31 shows that IL-23-induced acanthosis is significantly reduced inIL-22 deficient mice, as described in Example 20.

FIG. 32 shows that IL-22 deficiency had no effect on IL-12-inducedacanthosis, as described in Example 20.

FIG. 33 shows that IL-23 induces IL-22 production from variousIL-23-activated lymphocytes, as described in Example 21.

FIG. 34 shows that IL-22 is a new effector cytokine from the Th_(IL-17)lineage, as described in Example 22.

FIG. 35 shows that IL-22 and IL-17 are produced by the same Th lineage(Th_(IL-17)), as described in Example 22.

FIG. 36 shows that IL-23 stimulates IL-22 production from uponactivation of naïve T cells, as described in Example 22.

FIG. 37 shows that IL-19, IL-20, IL-22, and IL24 induce epidermalthickening, as described in Example 23.

FIG. 38 shows quantification of epidermal acanthosis induced by IL-19,IL-20, IL-22, and IL24, as described in Example 23.

FIG. 39 shows that components of the receptors for IL-19, IL-20, andIL-22 are expressed on human keratinocytes, as described in Example 24.

FIG. 40 shows that blocking antibodies to components of the receptorsfor IL-19, IL-20, and IL-22 reduce psoriasin expression, as described inExample 24.

FIG. 41 shows that antibodies to IL20Ra and IL-22R, when used incombination, effectively block IL-20-induced expression of psoriasin.

DETAILED DESCRIPTION OF EMBODIMENTS I. Definitions

The term “IL-22 polypeptide” or “IL-22” refers to various interleukin-22polypeptides (also referred to as “interleukin-22 ligand” or “IL-22L” inthe art). The term encompasses native sequence IL-22 polypeptides andvariants thereof (which are further defined herein). The IL-22polypeptides described herein may be isolated from a variety of sources,such as from human tissue or from another source, or prepared byrecombinant or synthetic methods. A native IL-22 may be from anyspecies, e.g., murine (“mIL-22”) or human (“hIL-22”).

The term “IL-22R polypeptide” or “IL-22R” refers to a polypeptidecomponent of an interleukin-22 receptor heterodimer or an interleukin-20receptor heterodimer. The term encompasses native sequence IL-22Rpolypeptides and variants thereof (which are further defined herein).The IL-22R polypeptides described herein may be isolated from a varietyof sources, such as from human tissue or from another source, orprepared by recombinant or synthetic methods. A native IL-22R may befrom any species, e.g., murine (“mIL-22R”) or human (“hIL-22R”). Nativesequence IL-22R polypeptides are also referred to in the art as“IL-22R1” and “IL22RA.”

A “native sequence IL-22 polypeptide” or a “native sequence IL-22Rpolypeptide” refers to a polypeptide comprising the same amino acidsequence as a corresponding IL-22 or IL-22R polypeptide derived fromnature. Such native sequence IL-22 or IL-22R polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The terms specifically encompass naturally-occurring truncated orsecreted forms of the specific IL-22 or IL-22R polypeptide (e.g., anIL-22 lacking its associated signal peptide), naturally-occurringvariant forms (e.g., alternatively spliced forms), andnaturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence IL-22 or IL-22Rpolypeptides disclosed herein are mature or full-length native sequencepolypeptides. FIGS. 2 and 3 show exemplary full length human IL-22 andIL-22R, respectively. A nucleic acid encoding the polypeptide shown inFIG. 2 is shown in FIG. 1. Start and stop codons are shown in bold fontand underlined in that figure. While the IL-22 and IL-22R polypeptidesequences disclosed in the accompanying figures are shown to begin withmethionine residues designated herein as amino acid position 1, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the IL-22 or IL-22Rpolypeptides.

An “IL-22 variant,” an “IL-22R variant,” an “IL-22 variant polypeptide,”or an “IL-22R variant polypeptide” means an active IL-22 or IL-22Rpolypeptide as defined above having at least about 80% amino acidsequence identity with a full-length native sequence IL-22 or IL-22Rpolypeptide sequence. Ordinarily, an IL-22 or IL-22R polypeptide variantwill have at least about 80% amino acid sequence identity, alternativelyat least about 81% amino acid sequence identity, alternatively at leastabout 82% amino acid sequence identity, alternatively at least about 83%amino acid sequence identity, alternatively at least about 84% aminoacid sequence identity, alternatively at least about 85% amino acidsequence identity, alternatively at least about 86% amino acid sequenceidentity, alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity, andalternatively at least about 99% amino acid sequence identity to afull-length or mature native sequence IL-22 or IL-22R polypeptidesequence.

“Percent (%) amino acid sequence identity,” with respect to the IL-22 orIL-22R polypeptide sequences identified herein, is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a specific IL-22 or IL-22Rpolypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Foramino acid sequence comparisons, the % amino acid sequence identity of agiven amino acid sequence A to, with, or against a given amino acidsequence B (which can alternatively be phrased as a given amino acidsequence A that has or comprises a certain % amino acid sequenceidentity to, with, or against a given amino acid sequence B) iscalculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program in that program's alignment of A andB, and where Y is the total number of amino acid residues in B. It willbe appreciated that where the length of amino acid sequence A is notequal to the length of amino acid sequence B, the % amino acid sequenceidentity of A to B will not equal the % amino acid sequence identity ofB to A. As examples of % amino acid sequence identity calculations usingthis method, Tables 2 and 3 demonstrate how to calculate the % aminoacid sequence identity of the amino acid sequence designated “ComparisonProtein” to the amino acid sequence designated “IL-22 or IL-22R”,wherein “IL-22 or IL-22R” represents the amino acid sequence of an IL-22or IL-22R polypeptide of interest, “Comparison Protein” represents theamino acid sequence of a polypeptide against which the “IL-22 or IL-22R”polypeptide of interest is being compared, and “X, “Y” and “Z” eachrepresent different amino acid residues.

TABLE 2 IL-22 or XXXXXXXXXXXXXXX (Length = 15 amino acids) IL-22RComparison XXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acidsequence identity = (the number of identically matching amino acidresidues between the two polypeptide sequences) divided by (the totalnumber of amino acid residues of the IL-22 or IL-22R polypeptide) = 5divided by 15 = 33.3%

TABLE 3 IL-22 or XXXXXXXXXX (Length = 10 amino acids) IL-22R ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences) divided by (the total number ofamino acid residues of the IL-22 or IL-22R polypeptide) = 5 divided by10 = 50%

The term “IL-19” refers to any native IL-19 from any vertebrate source,including mammals such as primates (e.g. humans and monkeys) and rodents(e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed IL-19 as well as any form of IL-19 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of IL-19, e.g., splice variants, allelic variants,and other isoforms. The term also encompasses fragments or variants of anative IL-19 that maintain at least one biological activity of IL-19.

The term “IL-20” refers to any native IL-20 from any vertebrate source,including mammals such as primates (e.g. humans and monkeys) and rodents(e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed IL-20 as well as any form of IL-20 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of IL-20, e.g., splice variants, allelic variants,and other isoforms. The term also encompasses fragments or variants of anative IL-20 that maintain at least one biological activity of IL-20.

The term “IL-24” refers to any native IL-24 from any vertebrate source,including mammals such as primates (e.g. humans and monkeys) and rodents(e.g., mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed IL-24 as well as any form of IL-24 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of IL-24, e.g., splice variants, allelic variants,and other isoforms. The term also encompasses fragments or variants of anative IL-24 that maintain at least one biological activity of IL-24.

The term “IL-22BP” or “IL-22 binding protein” as used herein refers toany native IL-22BP from any vertebrate source, including mammals such asprimates (e.g. humans and monkeys) and rodents (e.g., mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed IL-22BP as well as any form of IL-22BP that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of IL-22BP, e.g., splice variants, allelic variants, and otherisoforms. The term also encompasses fragments or variants of a nativeIL-22BP that maintain at least one biological activity of IL-22BP.Native IL-22BP is also referred to as “IL-22RA2” in the art.

The term IL-20Ra refers to a polypeptide component of an IL-19 receptorheterodimer or an IL-20 receptor heterodimer. The term encompasses anynative IL-20Ra from any vertebrate source, including mammals such asprimates (e.g. humans and monkeys) and rodents (e.g., mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed IL-20Ra as well as any form of IL-20Ra that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of IL-20Ra, e.g., splice variants, allelic variants, and otherisoforms. The term also encompasses fragments or variants of a nativeIL-20Ra that maintain at least one biological activity of IL-20Ra.Native IL-20Ra is also referred to as “IL-20R1” in the art.

The term IL-20Rb refers to a polypeptide component of an IL-19 receptorheterodimer or an IL-20 receptor heterodimer. The term encompasses anynative IL-20Rb from any vertebrate source, including mammals such asprimates (e.g. humans and monkeys) and rodents (e.g., mice and rats),unless otherwise indicated. The term encompasses “full-length,”unprocessed IL-20Rb as well as any form of IL-20Rb that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of IL-20Rb, e.g., splice variants, allelic variants, and otherisoforms. The term also encompasses fragments or variants of a nativeIL-20Rb that maintain at least one biological activity of IL-20Rb.Native IL-20Rb is also referred to as “IL-20R2” in the art.

The term “IL-10R2” refers to a polypeptide component of an IL-22receptor heterodimer or an IL-20 receptor heterodimer. The termencompasses any native IL-10R2 from any vertebrate source, includingmammals such as primates (e.g. humans and monkeys) and rodents (e.g.,mice and rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed IL-10R2 as well as any form of IL-10R2 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of IL-10R2, e.g., splice variants, allelic variants,and other isoforms. The term also encompasses fragments or variants of anative IL-10R2 that maintain at least one biological activity ofIL-10R2. Native IL-10R2 is also referred to as “IL-10Rb” in the art.

An “isolated” biological molecule, such as the various polypeptides,polynucleotides, and antibodies disclosed herein, refers to a biologicalmolecule that has been identified and separated and/or recovered from atleast one component of its natural environment.

“Active” or “activity,” with reference to IL-22 or IL-22R, refers to abiological and/or an immunological activity of a native IL-22 or IL-22R,wherein “biological” activity refers to a biological function of anative IL-22 or IL-22R other than the ability to induce the productionof an antibody against an antigenic epitope possessed by the nativeIL-22 or IL-22R. An “immunological” activity refers to the ability toinduce the production of an antibody against an antigenic epitopepossessed by a native IL-22 or IL-22R.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a polypeptide, such as a native IL-22 or IL-22Rpolypeptide. Also encompassed by “antagonist” are molecules that fullyor partially inhibit the transcription or translation of mRNA encodingthe polypeptide. Suitable antagonist molecules include, e.g., antagonistantibodies or antibody fragments; fragments or amino acid sequencevariants of a native polypeptide; peptides; antisense oligonucleotides;small organic molecules; and nucleic acids that encode polypeptideantagonists or antagonist antibodies. Reference to “an” antagonistencompasses a single antagonist or a combination of two or moredifferent antagonists.

The term “agonist” is used in the broadest sense and includes anymolecule that partially or fully mimics a biological activity of apolypeptide, such as a native IL-22 or IL-22R polypeptide. Alsoencompassed by “agonist” are molecules that stimulate the transcriptionor translation of mRNA encoding the polypeptide. Suitable agonistmolecules include, e.g., agonist antibodies or antibody fragments; anative polypeptide; fragments or amino acid sequence variants of anative polypeptide; peptides; antisense oligonucleotides; small organicmolecules; and nucleic acids that encode polypeptides agonists orantibodies. Reference to “an” agonist encompasses a single agonist or acombination of two or more different agonists.

“Alleviation” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of an agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, rodents (e.g., mice and rats), and monkeys;domestic and farm animals; and zoo, sports, laboratory, or pet animals,such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Insome embodiments, the mammal is selected from a human, rodent, ormonkey.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingsimilar structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, monovalentantibodies, multivalent antibodies, multispecific antibodies (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity) and may also include certain antibody fragments (as describedin greater detail herein). An antibody can be chimeric, human, humanizedand/or affinity matured.

An antibody that specifically binds to a particular antigen refers to anantibody that is capable of binding the antigen with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting the antigen. Preferably, the extent of binding ofsuch an antibody to a non-target polypeptide is less than about 10% ofthe binding of the antibody to the target antigen as measured, e.g., bya radioimmunoassay (RIA). In certain embodiments, an antibody that bindsto a target antigen has a dissociation constant (Kd) of ≦1 μM, ≦100 nM,≦10 nM, ≦1 nM, or ≦0.1 nM.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions (HVRs) both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in the binding of an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise on antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO93/1161; Hudson et al. (2003) Nat. Med 9:129-134; andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al. (2003)Nat. Med 9:129-134.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2^(nd) ed. 1988); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies(see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); andLee et al., J. Immunol. Methods 284(1-2): 119-132(2004), andtechnologies for producing human or human-like antibodies in animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO98/24893;WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad.Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Markset al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al.,Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93(1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies may be produced by procedures knownin the art. Marks et al. Bio/Technology 10:779-783 (1992) describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofHVR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody, “neutralizing” antibody, or “antagonist” antibodyis one which inhibits or reduces a biological activity of the antigen itbinds. Such antibodies may substantially or completely inhibit thebiological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which partially orfully mimics a biological activity of a polypeptide of interest.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol. 293:865-881).To establish conditions for the assay, microliter plates (Dynex) arecoated overnight with 5 μg/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of the anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays.

According to another embodiment, the Kd or Kd value is measured bysurface plasmon resonance assays using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on.) See, e.g.,Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If the on-rateexceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on)”according to this invention can also be determined as described aboveusing a BIAcore™-2000 or a BIAcore™-3000 system (BIAcore, Inc.,Piscataway, N.J.).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to a molecule(such as a nucleic acid, polypeptide, or antibody) so as to generate a“labeled” molecule. The label may be detectable by itself (e.g.radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition, resulting in a detectable product.

By “solid phase” is meant a non-aqueous matrix to which a molecule (suchas a nucleic acid, polypeptide, or antibody) can adhere. Examples ofsolid phases encompassed herein include those formed partially orentirely of glass (e.g., controlled pore glass), polysaccharides (e.g.,agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.In certain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a nucleic acid, polypeptide, antibody, agonist or antagonist)to a mammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

A “small molecule” or “small organic molecule” is defined herein as anorganic molecule having a molecular weight below about 500 Daltons.

An “oligopeptide” that binds to a target polypeptide is an oligopeptidethat is capable of binding the target polypeptide with sufficientaffinity such that the oligopeptide is useful as a diagnostic and/ortherapeutic agent in targeting the polypeptide. In certain embodiments,the extent of binding of an oligopeptide to an unrelated, non-targetpolypeptide is less than about 10% of the binding of the oligopeptide tothe target polypeptide as measured, e.g., by a surface plasmon resonanceassay. In certain embodiments, an oligopeptide binds to a targetpolypeptide with a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM,≦1 nM, or ≦0.1 nM.

An “organic molecule” that binds to a target polypeptide is an organicmolecule other than an oligopeptide or antibody as defined herein thatis capable of binding a target polypeptide with sufficient affinity suchthat the organic molecule is useful as a diagnostic and/or therapeuticagent in targeting the polypeptide. In certain embodiments, the extentof binding of an organic molecule to an unrelated, non-targetpolypeptide is less than about 10% of the binding of the organicmolecule to the target polypeptide as measured, e.g., by a surfaceplasmon resonance assay. In certain embodiments, an organic moleculebinds to a target polypeptide with a dissociation constant (Kd) of ≦1μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM.

A “biological system” is an in vitro, ex vivo, or in vivo systemcomprising mammalian cells that share a common signaling pathway.

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,and neoplasia.

The term “T cell mediated disease” means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to a morbidity ina mammal. The T cell mediated disease may be associated with cellmediated effects, lymphokine mediated effects, etc., and even effectsassociated with B cells if the B cells are stimulated, for example, bythe lymphokines secreted by T cells.

As used herein the term “psoriasis” is defined as a conditioncharacterized by the eruption of circumscribed, discreet and confluent,reddish, silvery-scaled macropapules preeminently on the elbows, knees,scalp or trunk.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The term “tumor progression” refers to the growth and/or proliferationof a tumor.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, rectal cancer, gastric cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, leukemia and other lymphoproliferative disorders, andvarious types of head and neck cancer.

An “autoimmune disorder” or “autoimmunity” refers to any condition inwhich a humoral or cell-mediated immune response is mounted against abody's own tissue. An “IL-23 mediated autoimmune disorder” is anyautoimmune disorder that is caused by, maintained, or exacerbated byIL-23 activity.

“Inflammation” refers to the accumulation of leukocytes and the dilationof blood vessels at a site of injury or infection, typically causingpain, swelling, and redness.

“Chronic inflammation” refers to inflammation in which the cause of theinflammation persists and is difficult or impossible to remove.

“Autoimmune inflammation” refers to inflammation associated with anautoimmune disorder.

“Arthritic inflammation” refers to inflammation associated witharthritis.

“Inflammatory bowel disease” or “IBD” refers to a chronic disordercharacterized by inflammation of the gastrointestinal tract. IBDencompasses ulcerative colitis, which affects the large intestine and/orrectum, and Crohn's disease, which may affect the entiregastrointestinal system but more commonly affects the small intestine(ileum) and possibly the large intestine.

“Arthritis” refers to inflammation of the joints and includes, but isnot limited to, osteoarthritis, gout, infection-associated arthritis,Reiter's syndrome arthritis, and arthritis associated with autoimmunedisorders, such as rheumatoid arthritis, psoriatic arthritis,lupus-associated arthritis, spondyloarthritis, andscleroderma-associated arthritis.

The term “effective amount” is a concentration or amount of a molecule(e.g., a nucleic acid, polypeptide, agonist, or antagonist) that resultsin achieving a particular stated purpose. An “effective amount” may bedetermined empirically. A “therapeutically effective amount” is aconcentration or amount of a molecule which is effective for achieving astated therapeutic effect. This amount may also be determinedempirically.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeadriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosinearabinoside (“Ara-C”), cyclophosphamide, thiotepa, busulfan, cytoxin,taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb Oncology,Princeton, N.J.), and doxetaxel (Taxotere, Rhône-Poulenc Rorer, Antony,France), toxotere, methotrexate, cisplatin, melphalan, vinblastine,bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,vincristine, vinorelbine, carboplatin, teniposide, daunomycin,carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins (seeU.S. Pat. No. 4,675,187), melphalan and other related nitrogen mustards.Also included in this definition are hormonal agents that act toregulate or inhibit hormone action on tumors such as tamoxifen andonapristone.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a cancer celloverexpressing any of the genes identified herein, either in vitro or invivo. Thus, a growth inhibitory agent is one which significantly reducesthe percentage of cells overexpressing such genes in S phase. Examplesof growth inhibitory agents include agents that block cell cycleprogression (at a place other than S phase), such as agents that induceGI arrest and M-phase arrest. Classical M-phase blockers include thevincas (vincristine and vinblastine), taxol, and topo II inhibitors suchas doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest GI also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al. (W BSaunders: Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell population as intercellularmediators. Examples of such cytokines are lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

As used herein, the term “inflammatory cells” designates cells thatenhance the inflammatory response such as mononuclear cells,eosinophils, macrophages, and polymorphonuclear neutrophils (PMN).

II. Compositions and Methods of the Invention

A. IL-22 or IL-22R Polynucleotides and Polypeptides

The present invention provides isolated IL-22 or IL-22R polypeptides andisolated nucleotide sequences encoding those polypeptides. IL-22 orIL-22R polypeptides encompass native full-length or mature IL-22 orIL-22R polypeptides as well as IL-22 or IL-22R variants IL-22 or IL-22Rvariants can be prepared by introducing appropriate nucleotide changesinto the IL-22 or IL-22R DNA, and/or by synthesis of the desired IL-22or IL-22R polypeptide. Those skilled in the art will appreciate thatamino acid changes may alter post-translational processing of the IL-22or IL-22R, such as changing the number or position of glycosylationsites or altering the membrane anchoring characteristics.

Variations in native IL-22 or IL-22R or in various domains of IL-22 orIL-22R, as described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the IL-22 or IL-22R that results in a change in theamino acid sequence of the IL-22 or IL-22R as compared with a nativesequence IL-22 or IL-22R. Optionally, the variation is by substitutionof at least one amino acid with any other amino acid in one or moredomains of the IL-22 or IL-22R. Guidance in determining which amino acidresidue may be inserted, substituted or deleted without adverselyaffecting the desired activity may be found by comparing the sequence ofthe IL-22 or IL-22R with that of homologous known protein molecules andminimizing the number of amino acid sequence changes made in regions ofhigh homology. Amino acid substitutions can be the result of replacingone amino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of about 1 to 5 amino acids. The variationallowed may be determined by systematically making insertions, deletionsor substitutions of amino acids in the sequence and testing theresulting variants for activity exhibited by the full-length or maturenative sequence.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N)gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asnGlu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg argIle (I) leu; val; met; ala; phe; leu norleucine Leu (L)norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn argMet (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P)ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y)trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of theIL-22 or IL-22R polypeptide are accomplished by selecting substitutionsthat differ significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain. Naturally occurring residues are divided into groupsbased on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon cloned DNA to produce an IL-22 or IL-22R variant DNA.

IL-22 or IL-22R polypeptide fragments are also provided herein. Suchfragments may be truncated at the N-terminus or C-terminus, or may lackinternal residues, for example, when compared with a full length nativeprotein. Certain fragments lack amino acid residues that are notessential for a desired biological activity of the IL-22 or IL-22Rpolypeptide. Accordingly, in certain embodiments, a fragment of IL-22 orIL-22R is biologically active. In certain embodiments, a fragment offull length IL-22 lacks the N-terminal signal peptide sequence. Incertain embodiments, a fragment of full-length IL-22R is a soluble formof IL-22R that is not membrane bound, e.g., a form of IL-22R that lacksa transmembrane domain. For example, a soluble form of human IL-22R maylack all or a substantial portion of the transmembrane domain from aboutamino acids 229-251 of SEQ ID NO:3.

Covalent modifications of IL-22 or IL-22R are included within the scopeof this invention. One type of covalent modification includes reactingtargeted amino acid residues of an IL-22 or IL-22R polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of the IL-22 or IL-22R.Derivatization with bifunctional agents is useful, for instance, forcrosslinking IL-22 or IL-22R to a water-insoluble support matrix orsurface for use in the method for purifying anti-IL-22 or IL-22Rantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of an IL-22 or IL-22R polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence IL-22 orIL-22R (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that are not present in thenative sequence IL-22 or IL-22R. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

An IL-22 or IL-22R polypeptide of the present invention may also bemodified in a way to form a chimeric molecule comprising IL-22 or IL-22Rfused to another, heterologous polypeptide or amino acid sequence. Inone embodiment, a chimeric molecule comprises a fusion of the IL-22 orIL-22R with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the IL-22 or IL-22R. Thepresence of such epitope-tagged forms of the IL-22 or IL-22R can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the IL-22 or IL-22R to be readily purified byaffinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. Various tag polypeptidesand their respective antibodies are well known in the art. Examplesinclude poly-histidine (poly-his) or poly-histidine-glycine(poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5[Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptidesinclude the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194(1992)]; an alpha-tubulin epitope peptide [Skinner et al., J. Biol.Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)].

In another embodiment, a chimeric molecule may comprise a fusion of anIL-22 or IL-22R polypeptide with an immunoglobulin or a particularregion of an immunoglobulin. For a bivalent form of the chimericmolecule (also referred to as an “immunoadhesin”), such a fusion couldbe to the Fc region of an IgG molecule. The Ig fusions preferablyinclude the substitution of a soluble form of an IL-22 or IL-22Rpolypeptide in place of at least one variable region within an Igmolecule. In a particularly preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgG1 molecule. For the production of immunoglobulinfusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

1. Preparation of IL-22 or IL-22R

IL-22 or IL-22R may be prepared by routine recombinant methods, e.g.,culturing cells transformed or transfected with a vector containing anucleic acid encoding an IL-22 or IL-22R, as exemplified by the nucleicacid shown in FIG. 1, which encodes an IL-22. Host cells comprising anysuch vector are also provided. By way of example, host cells may be CHOcells, E. coli, or yeast. A process for producing any of the hereindescribed polypeptides is further provided and comprises culturing hostcells under conditions suitable for expression of the desiredpolypeptide and recovering the desired polypeptide from the cellculture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or an Fc region of an immunoglobulin.

It is, of course, contemplated that alternative methods, which are wellknown in the art, may be employed to prepare IL-22 or IL-22R. Forinstance, the IL-22 or IL-22R sequence, or portions thereof, may beproduced by direct peptide synthesis using solid-phase techniques [see,e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co.,San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the IL-22 or IL-22R may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe full-length IL-22 or IL-22R.

Recombinantly expressed IL-22 or IL-22R may be recovered from culturemedium or from host cell lysates. The following procedures are exemplaryof suitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the IL-22 or IL-22R. Various methods of proteinpurification may be employed and such methods are known in the art anddescribed for example in Deutscher, Methods in Enzymology, 182 (1990);Scopes, Protein Purification: Principles and Practice, Springer-Verlag,New York (1982). The purification step(s) selected will depend, forexample, on the nature of the production process used and the particularIL-22 or IL-22R produced.

2. Detection of Gene Expression

Expression of a gene encoding IL-22 or IL-22R can be detected by variousmethods in the art, e.g, by detecting expression of mRNA encoding IL-22or IL-22R. As used herein, the term “detecting” encompasses quantitativeor qualitative detection. By detecting IL-22 or IL-22R gene expression,one can identify, e.g., those tissues that express an IL-22 or IL-22Rgene. Gene expression may be measured using certain methods known tothose skilled in the art, e.g., Northern blotting, (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 [1980]); quantitative PCR; or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, gene expression may bemeasured by immunological methods, such as immunohistochemical stainingof tissue sections and assay of cell culture or body fluids, toquantitate directly the expression of gene product. Antibodies usefulfor immunohistochemical staining and/or assay of sample fluids encompassany of the antibodies provided herein. Conveniently, the antibodies maybe prepared against a native sequence of an IL-22 or IL-22R polypeptide;against a synthetic peptide comprising a fragment of an IL-22 or IL-22Rpolypeptide sequence; or against an exogenous sequence fused to an IL-22or IL-22R polypeptide or fragment thereof (including a syntheticpeptide).

B. Antibodies

Antibodies that bind to any of the above- or below-describedpolypeptides are provided. In one embodiment, an isolated antibody thatbinds to an IL-19, IL-20, IL-22, IL-24, IL-20Ra, IL-20Rb, IL-10R2, orIL-22R polypeptide. Exemplary antibodies include polyclonal, monoclonal,humanized, human, bispecific, and heteroconjugate antibodies. Anantibody may be an antibody fragment, e.g., a Fab, Fab′-SH, Fv, scFv, or(Fab′)2 fragment. In one embodiment, an isolated antibody that binds toan IL-22 or IL-22R is provided. In one such embodiment, an antibodypartially or completely blocks the activity of an IL-22 or IL-22Rpolypeptide (i.e., a “blocking” antibody).

Exemplary monoclonal antibodies that bind IL-22 and IL-22R are providedherein and are further described in the Examples. Those antibodiesinclude the anti-IL-22 antibodies designated 3F11.3 (“3F11”), 11H4.4(“11H4”), and 8E11.9 (“8E11”), and the anti-IL-22R antibodies designated7E9.10.8 (“7E9”), 8A12.32 (“8A12”), 8H11.32.28 (“8H11”), and 12H5. Inone embodiment, a hybridoma that produces any of those antibodies isprovided. In one embodiment, monoclonal antibodies that compete with3F11.3, 11H4.4, or 8E11.9 for binding to IL-22 are provided. In anotherembodiment, monoclonal antibodies that bind to the same epitope as3F11.3, 11H4.4, or 8E11.9 are provided. In another embodiment,monoclonal antibodies that compete with 7E9, 8A12, 8H11, or 12H5 forbinding to IL-22R are provided. In one embodiment, monoclonal antibodiesthat bind to the same epitope as 7E9, 8A12, 8H11, or 12H5 are provided.Various embodiments of antibodies are provided below:

1. Polyclonal Antibodies

Antibodies may comprise polyclonal antibodies. Methods of preparingpolyclonal antibodies are known to the skilled artisan. Polyclonalantibodies can be raised in a mammal, for example, by one or moreinjections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the polypeptide of interest or a fusionprotein thereof. It may be useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include but are not limited to keyholelimpet hemocyanin, serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. Examples of adjuvants which may be employed includeFreund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

Antibodies may, alternatively, be monoclonal antibodies. Monoclonalantibodies may be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The immunizing agent will typically include the polypeptide of interestor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies that bind to thepolypeptide of interest. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can be made by using combinatorial libraries toscreen for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are describedgenerally in Hoogenboom et al. (2001) in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J.), and incertain embodiments, in Lee et al. (2004) J. Mol. Biol. 340:1073-1093.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the antibodies of the invention can beobtained by designing a suitable antigen screening procedure to selectfor the phage clone of interest followed by construction of a fulllength antibody clone using the Fv sequences from the phage clone ofinterest and suitable constant region (Fc) sequences described in Kabatet al., Sequences of Proteins of Immunological Interest, Fifth Edition,NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

3. Monovalent Antibodies

Monovalent antibodies are also provided. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

4. Antibody Fragments

Antibody fragments are also provided. Antibody fragments may begenerated by traditional means, such as enzymatic digestion, or byrecombinant techniques. In certain circumstances there are advantages ofusing antibody fragments, rather than whole antibodies. The smaller sizeof the fragments allows for rapid clearance, and may lead to improvedaccess to solid tumors. For a review of certain antibody fragments, seeHudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

5. Humanized Antibodies

Humanized antibodies are also provided. Various methods for humanizingnon-human antibodies are known in the art. For example, a humanizedantibody can have one or more amino acid residues introduced into itfrom a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

6. Human Antibodies

Human antibodies are also provided. Human antibodies can be constructedby combining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s) as described above. Alternatively, human monoclonalantibodies of the invention can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by KozborJ. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255(1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

7. Bispecific Antibodies

Bispecific antibodies are also provided. Bispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent antigens. In certain embodiments, bispecific antibodies arehuman or humanized antibodies. In certain embodiments, one of thebinding specificities is for a polypeptide of interest and the other isfor any other antigen. In certain embodiments, bispecific antibodies maybind to two different epitopes of a polypeptide of interest. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress a polypeptide of interest, such a cell surface polypeptide.These antibodies possess a TAT226-binding arm and an arm which binds acytotoxic agent, such as, e.g., saporin, anti-interferon-α, vincaalkaloid, ricin A chain, methotrexate or radioactive isotope hapten.Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking method. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

8. Multivalent Antibodies

Multivalent antibodies are also provided. A multivalent antibody may beinternalized (and/or catabolized) faster than a bivalent antibody by acell expressing an antigen to which the antibodies bind. The antibodiesof the present invention can be multivalent antibodies (which are otherthan of the IgM class) with three or more antigen binding sites (e.g.tetravalent antibodies), which can be readily produced by recombinantexpression of nucleic acid encoding the polypeptide chains of theantibody. The multivalent antibody can comprise a dimerization domainand three or more antigen binding sites. In certain embodiments, thedimerization domain comprises (or consists of) an Fc region or a hingeregion. In this scenario, the antibody will comprise an Fc region andthree or more antigen binding sites amino-terminal to the Fc region. Incertain embodiments, a multivalent antibody comprises (or consists of)three to about eight antigen binding sites. In one such embodiment, amultivalent antibody comprises (or consists of) four antigen bindingsites. The multivalent antibody comprises at least one polypeptide chain(for example, two polypeptide chains), wherein the polypeptide chain(s)comprise two or more variable domains. For instance, the polypeptidechain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a firstvariable domain, VD2 is a second variable domain, Fc is one polypeptidechain of an Fc region, X1 and X2 represent an amino acid or polypeptide,and n is 0 or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (for example, four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

9. Single-Domain Antibodies

Single-domain antibodies are also provided. A single-domain antibody isa single polypeptide chain comprising all or a portion of the heavychain variable domain or all or a portion of the light chain variabledomain of an antibody. In certain embodiments, a single-domain antibodyis a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see,e.g., U.S. Pat. No. 6,248,516 B1). In one embodiment, a single-domainantibody consists of all or a portion of the heavy chain variable domainof an antibody.

10. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

In certain embodiments, a glycosylation variant comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. Sites of interest for substitutionalmutagenesis include the hypervariable regions, but FR alterations arealso contemplated. Conservative substitutions are shown in Table 6 aboveunder the heading of “preferred substitutions.” If such substitutionsresult in a desirable change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 6,or as further described above in reference to amino acid classes, may beintroduced and the resulting antibodies screened for the desired bindingproperties.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibodies thusgenerated are displayed from filamentous phage particles as fusions toat least part of a phage coat protein (e.g., the gene III product ofM13) packaged within each particle. The phage-displayed variants arethen screened for their biological activity (e.g. binding affinity). Inorder to identify candidate hypervariable region sites for modification,scanning mutagenesis (e.g., alanine scanning) can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according totechniques known in the art, including those elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening using techniques known in the art, including those describedherein, and antibodies with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

In one aspect, the invention provides antibodies comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

11. Antibody Derivatives

Antibodies can be further modified to contain additionalnonproteinaceous moieties that are known in the art and readilyavailable. Preferably, the moieties suitable for derivatization of theantibody are water soluble polymers. Non-limiting examples of watersoluble polymers include, but are not limited to, polyethylene glycol(PEG), copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)).The radiation may be of any wavelength, and includes, but is not limitedto, wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theantibody-nonproteinaceous moiety are killed.

In certain embodiments, an antibody may be labeled and/or may beimmobilized on a solid support. In a further aspect, an antibody is ananti-idiotypic antibody.

12. Heteroconjugate Antibodies

Heteroconjugate antibodies are also provided. Heteroconjugate antibodiesare composed of two covalently joined antibodies. Such antibodies have,for example, been proposed to target immune system cells to unwantedcells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO91/00360; WO 92/200373; EP 03089]. It is contemplated that theantibodies may be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins may be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsfor this purpose include iminothiolate and methyl-4-mercaptobutyrimidateand those disclosed, for example, in U.S. Pat. No. 4,676,980.

13. Cytotoxic Antibodies

Cytotoxic antibodies are also provided. In certain embodiments, acytotoxic antibody is an anti-IL22 antibody, such as those providedbelow, which effects an effector function and/or induces cell death. Incertain embodiments, a cytotoxic anti-IL-22R antibody binds to theextracellular domain of an IL-22R.

14. Effector Function Engineering

It may be desirable to modify an antibody with respect to effectorfunction, so as to enhance, e.g., the effectiveness of the antibody intreating a disease such as cancer. For example, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

15. Vectors, Host Cells, and Recombinant Methods

For recombinant production of an antibody, in one embodiment, thenucleic acid encoding it is isolated and inserted into a replicablevector for further cloning (amplification of the DNA) or for expression.DNA encoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, hostcells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a) Generating Antibodies Using Prokaryotic Host Cells (1) VectorConstruction

Polynucleotide sequences encoding polypeptide components of an antibodycan be obtained using standard recombinant techniques. Desiredpolynucleotide sequences may be isolated and sequenced from antibodyproducing cells such as hybridoma cells. Alternatively, polynucleotidescan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in prokaryotic hosts. Many vectors that are availableand known in the art can be used for the purpose of the presentinvention. Selection of an appropriate vector will depend mainly on thesize of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell may be usedin connection with these hosts. The vector ordinarily carries areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

An expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies of the invention. Such modulation isaccomplished at least in part by simultaneously modulating translationalstrengths for the polypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence. In certain embodiments, changes in the nucleotidesequence are silent. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

In one embodiment, a set of vectors is generated with a range of TIRstrengths for each cistron therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofthe desired antibody products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. Based on the translational strength comparison, the desiredindividual TIRs are selected to be combined in the expression vectorconstructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coliλ 1776 (ATCC31,537) and E. coli RV308(ATCC 31,608) are also suitable. These examplesare illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

(2) Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycolate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. Incertain embodiments, for E. coli growth, growth temperatures range fromabout 20° C. to about 39° C.; from about 25° C. to about 37° C.; orabout 30° C. The pH of the medium may be any pH ranging from about 5 toabout 9, depending mainly on the host organism. In certain embodiments,for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. In certain embodiments, thephosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, and in certain embodiments, about 1,000 to 100,000 liters ofcapacity. These fermentors use agitator impellers to distribute oxygenand nutrients, especially glucose (the preferred carbon/energy source).Small scale fermentation refers generally to fermentation in a fermentorthat is no more than approximately 100 liters in volumetric capacity,and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins: suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

(3) Antibody Purification

In one embodiment, an antibody produced herein is further purified toobtain preparations that are substantially homogeneous for furtherassays and uses. Standard protein purification methods known in the artcan be employed. The following procedures are exemplary of suitablepurification procedures: fractionation on immunoaffinity or ion-exchangecolumns, ethanol precipitation, reverse phase HPLC, chromatography onsilica or on a cation-exchange resin such as DEAE, chromatofocusing,SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, forexample, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized can be a column comprising a glass or silica surface, ora controlled pore glass column or a silicic acid column. In someapplications, the column is coated with a reagent, such as glycerol, topossibly prevent nonspecific adherence of contaminants.

As the first step of purification, a preparation derived from the cellculture as described above can be applied onto a Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase would then be washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b) Generating Antibodies Using Eukaryotic Host Cells

A vector for use in a eukaryotic host cell generally includes one ormore of the following non-limiting components: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(1) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected may be one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such a precursor region is ligated in readingframe to DNA encoding the antibody.

(2) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(3) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, in some embodiments, cells transformed with the DHFRselection gene are first identified by culturing all of thetransformants in a culture medium that contains methotrexate (Mtx), acompetitive antagonist of DHFR. In some embodiments, an appropriate hostcell when wild-type DHFR is employed is the Chinese hamster ovary (CHO)cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(4) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a polypeptide of interest (e.g., an antibody). Promotersequences are known for eukaryotes. For example, virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. In certain embodiments, any or all of these sequences may besuitably inserted into eukaryotic expression vectors.

Transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describingexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

Transcription of DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) describing enhancerelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibodypolypeptide-encoding sequence, but is generally located at a site 5′from the promoter.

(6) Transcription Termination Component

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(7) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(8) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othersupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art. The culture conditions, suchas temperature, pH, and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

(9) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, may be removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems may be firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis, and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a convenient technique. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody. Protein A can be used topurify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached may be agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to furtherpurification, for example, by low pH hydrophobic interactionchromatography using an elution buffer at a pH between about 2.5-4.5,preferably performed at low salt concentrations (e.g., from about0-0.25M salt).

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical use are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

C. Immunoconjugates

Immunoconjugates, or “antibody-drug conjugates,” are useful for thelocal delivery of cytotoxic agents in the treatment of cancer. See,e.g., Syrigos et al. (1999) Anticancer Research 19:605-614;Niculescu-Duvaz et al. (1997) Adv. Drug Deliv. Rev. 26:151-172; U.S.Pat. No. 4,975,278. Immunoconjugates allow for the targeted delivery ofa drug moiety to a tumor, whereas systemic administration ofunconjugated cytotoxic agents may result in unacceptable levels oftoxicity to normal cells as well as the tumor cells sought to beeliminated. See Baldwin et al. (Mar. 15, 1986) Lancet pp. 603-05; Thorpe(1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological and ClinicalApplications (A. Pinchera et al., eds.) pp. 475-506.

In one aspect, an immunoconjugate comprises an antibody that bindsIL-19, IL-20, IL-22, IL-24, IL22R, IL-20Ra, IL-20Rb, or IL-10R2, such asthose provided herein, and a cytotoxic agent, such as a chemotherapeuticagent, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

1. Maytansine and Maytansinoids

In one embodiment, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are mitototicinhibitors which act by inhibiting tubulin polymerization. Maytansinewas first isolated from the east African shrub Maytenus serrata (U.S.Pat. No. 3,896,111). Subsequently, it was discovered that certainmicrobes also produce maytansinoids, ids, such as maytansinol and C-3maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol andderivatives and analogues thereof are disclosed, for example, in U.S.Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946;4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866;4,424,219; 4,450,254; 4,362,663; and 4,371,533, the disclosures of whichare hereby expressly incorporated by reference.

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies that bind to antigenson the surface of tumor cells. Immunoconjugates containing maytansinoidsand their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describedimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansonid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. An average of 3-4 maytansinoid molecules conjugated perantibody molecule has shown efficacy in enhancing cytotoxicity of targetcells without negatively affecting the function or solubility of theantibody, although even one molecule of toxin per antibody would beexpected to enhance cytotoxicity over the use of naked antibody.Maytansinoids are well known in the art and can be synthesized usingknown techniques or isolated from natural sources. Suitablemaytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 andin the other patents and nonpatent publications referred to hereinabove.Preferred maytansinoids are maytansinol and maytansinol analoguesmodified in the aromatic ring or at other positions of the maytansinolmolecule, such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Certain coupling agents, includingN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al.,Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhyrdoxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

2. Auristatins and Dolastatins

In some embodiments, an immunoconjugate comprises an antibody conjugatedto a dolastatin or dolostatin peptidic analog or derivative, e.g., anauristatin (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin orauristatin drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands,” USPatent Application Publication No. US 2005-0238649 A1, the disclosure ofwhich is expressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat. Biotechnol.21(7):778-784; US Patent Application Publication No. 2005-0238649 A1,hereby incorporated by reference in its entirety (disclosing, e.g.,linkers and methods of preparing monomethylvaline compounds such as MMAEand MMAF conjugated to linkers).

3. Calicheamicin

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. For the preparation of conjugates of thecalicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all toAmerican Cyanamid Company). Structural analogues of calicheamicin whichmay be used include, but are not limited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I),N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug to which the antibody can be conjugated is QFA which isan antifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

4. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to an antibody includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively as LL-E33288 complex described in U.S. Pat.Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

In another aspect, an immunoconjugate may comprise an antibody and acompound with nucleolytic activity (e.g., a ribonuclease or a DNAendonuclease such as a deoxyribonuclease; DNase).

For selective destruction of a tumor, an immunoconjugate may comprise ananti-FGFR2 antibody and a highly radioactive atom. A variety ofradioactive isotopes are available for the production of radioconjugatedanti-FGFR2 antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶,Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When theconjugate is used for diagnosis, it may comprise a radioactive atom forscintigraphic studies, for example tc^(99m) or I¹²³, or a spin label fornuclear magnetic resonance (NMR) imaging (also known as magneticresonance imaging, mri), such as iodine-123 again, iodine-131,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron.

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

D. Antagonists and Agonists

Antagonists of IL-22 are provided. Such antagonists encompass those thatdirectly act on IL-22 (e.g., an anti-IL-22 antibody) and those thatindirectly affect IL-22 activity (e.g., an anti-IL-22R antibody). Suchantagonists are useful, for example, for 1) treating inflammatorydisorders and autoimmune disorders, and 2) modulating IL-23 or IL-22signaling In one particular embodiment, a composition comprising anantagonist of IL-22 or IL-22R is useful for reducing the amount ofpsoriatic tissue in a mammal. In another particular embodiment, acomposition comprising an antagonist of IL-22 or IL-22R is useful forpartially or fully inhibiting tumor cell proliferation.

In one aspect, an antagonist of IL-22 is an anti-IL-22 antibody or ananti-IL-22R antibody. In certain embodiments, an anti-IL-22 antibody isa blocking antibody that fully or partially blocks the interaction ofIL-22 with its receptor. In certain embodiments, an anti-IL-22R antibodyis a blocking antibody that fully or partially blocks the interaction ofIL-22R with IL-22. In certain embodiments, an anti-IL-22R antibody bindsto the extracellular ligand binding domain of an IL-22R. For example, ananti-IL-22R antibody may bind to the extracellular ligand binding domainof human IL-22R, which is found in SEQ ID NO:3 from about amino acids18-228.

In another aspect, an antagonist of IL-22 is an oligopeptide that bindsto IL-22 or IL-22R. In one embodiment, an oligopeptide binds to theextracellular ligand binding domain of IL-22R. Oligopeptides may bechemically synthesized using known oligopeptide synthesis methodology ormay be prepared and purified using recombinant technology. Sucholigopeptides are usually at least about 5 amino acids in length,alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length.Such oligopeptides may be identified without undue experimentation usingwell known techniques. In this regard, it is noted that techniques forscreening oligopeptide libraries for oligopeptides that are capable ofspecifically binding to a polypeptide target are well known in the art(see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092,5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A.,81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA,82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens,130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987);Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al.(1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991)Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624;Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al.(1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)Current Opin. Biotechnol., 2:668). In certain embodiments, anoligopeptide may be conjugated to a cytotoxic agent.

In yet another aspect, an antagonist of IL-22 is an organic moleculethat binds to IL-22 or IL-22R, other than an oligopeptide or antibody asdescribed herein. An organic molecule may be, for example, a smallmolecule. In one embodiment, an organic molecule binds to theextracellular domain of an IL-22R. An organic molecule that binds toIL-22 or IL-22R may be identified and chemically synthesized using knownmethodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).Such organic molecules are usually less than about 2000 daltons in size,alternatively less than about 1500, 750, 500, 250 or 200 daltons insize, wherein such organic molecules that are capable of binding toIL-22 or IL-22R may be identified without undue experimentation usingwell known techniques. In this regard, it is noted that techniques forscreening organic molecule libraries for molecules that are capable ofbinding to a polypeptide target are well known in the art (see, e.g.,PCT Publication Nos. WO00/00823 and WO00/39585). In certain embodiments,an organic molecule may be conjugated to a cytotoxic agent.

In yet another aspect, an IL-22 antagonist is a soluble IL-22 receptor,e.g., a form of IL-22R that is not membrane bound. Such soluble forms ofIL-22R may compete with membrane-bound IL-22R for binding to IL-22. Incertain embodiments, a soluble form of IL-22R may comprise all or aligand-binding portion of an extracellular domain of IL-22R, e.g., allor a ligand-binding portion of a polypeptide comprising amino acids18-228 of SEQ ID NO:3. In certain embodiments, a soluble form of IL-22Rlacks a transmembrane domain. For example, a soluble form of humanIL-22R may lack all or a substantial portion of the transmembrane domainfrom about amino acids 229-251 of SEQ ID NO:3.

A naturally occurring, soluble receptor for IL-22 has been reported. SeeDumoutier L. et al., “Cloning and characterization of IL-22 bindingprotein, a natural antagonist of IL-10-related T cell-derived induciblefactor/IL-22,” J. Immunol, 166:7090-7095 (2001); and Xu W. et al., “Asoluble class II cytokine receptor, IL-22RA2, is a naturally occurringIL-22 antagonist,” Proc. Natl. Acad. Sci. U.S.A. 98:9511-9516 (2001).That receptor is variously designated “IL-22BP” or “IL-22RA2” in theart. The sequence of a human IL-22BP is shown in FIG. 4. The term“IL-22BP” or “IL-22 binding protein” as used herein refers to any nativeIL-22BP from any vertebrate source, including mammals such as primates(e.g. humans and monkeys) and rodents (e.g., mice and rats), unlessotherwise indicated.

In yet another aspect, an antagonist of IL-22 is an antisense nucleicacid that decreases expression of the IL-22 or IL-22R gene (i.e., thatdecreases transcription of the IL-22 or IL-22R gene and/or translationof IL-22 or IL-22R mRNA). In certain embodiments, an antisense nucleicacid binds to a nucleic acid (DNA or RNA) encoding IL-22 or IL-22R. Incertain embodiments, an antisense nucleic acid is an oligonucleotide ofabout 10-30 nucleotides in length (including all points between thoseendpoints). In certain embodiments, an antisense oligonucleotidecomprises a modified sugar-phosphodiester backbones (or other sugarlinkages, including phosphorothioate linkages and linkages as describedin WO 91/06629), wherein such modified sugar-phosphodiester backbonesare resistant to endogenous nucleases. In one embodiment, an antisensenucleic acid is an oligodeoxyribonucleotide, which results in thedegradation and/or reduced transcription or translation of mRNA encodingIL-22 or IL-22R. In certain embodiments, an antisense nucleic acid is anRNA that reduces expression of a target nucleic acid by “RNAinterference” (“RNAi”). For review of RNAi, see, e.g., Novina et al.(2004) Nature 430:161-164. Such RNAs are derived from, for example,short interfering RNAs (siRNAs) and microRNAs. siRNAs, e.g., may besynthesized as double stranded oligoribonucleotides of about 18-26nucleotides in length. Id.

In yet another aspect, agonists of IL-22 are provided. Exemplaryagonists include, but are not limited to, native IL-22 or IL-22R;fragments, variants, or modified forms of IL-22 or IL-22R that retain atleast one activity of the native polypeptide; agents that are able tobind to and activate IL-22R; and agents that induce overexpression ofIL-22 or IL-22R or nucleic acids encoding IL-22 or IL-22R.

E. Pharmaceutical Formulations

The invention provides pharmaceutical formulations. In one embodiment, apharmaceutical formulation comprises 1) an active agent, e.g., any ofthe above-described polypeptides, antibodies, agonists, or antagonists;and 2) a pharmaceutically acceptable carrier. In a further embodiment, apharmaceutical formulation further comprises at least one additionaltherapeutic agent.

Pharmaceutical formulations are prepared for storage by mixing an agenthaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Lipofections or liposomes can also be used to deliver an agent into acell. Where the agent is an antibody fragment, the smallest inhibitoryfragment which specifically binds to the target protein is preferred.For example, based upon the variable region sequences of an antibody,peptide molecules can be designed which retain the ability to bind thetarget protein sequence. Such peptides can be synthesized chemicallyand/or produced by recombinant DNA technology (see, e.g., Marasco etal., Proc. Natl. Acad. Sci. USA 90, 7889-7893 [1993]). Antibodiesdisclosed herein may also be formulated as immunoliposomes. Liposomescontaining an antibody are prepared by methods known in the art, such asdescribed in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688(1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); andU.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Particularlyuseful liposomes can be generated by the reverse-phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. Fab′ fragments of an antibody ofthe present invention can be conjugated to liposomes as described inMartin et al., J. Biol. Chem., 257: 286-288 (1982) via adisulfide-interchange reaction. A chemotherapeutic agent (such asdoxorubicin) is optionally contained within the liposome. See Gabizon etal., J. National Cancer Inst., 81(19): 1484 (1989).

An agent may also be entrapped in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of an agent may be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the agent, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

A pharmaceutical formulation herein may also contain more than oneactive compound as necessary for the particular indication beingtreated. For example, in one aspect, a pharmaceutical formulationcontaining more than one active compound comprises 1) at least oneantagonist of IL-22, e.g., an antibody that binds to IL-22 and/or anantibody that binds to IL-22R; and 2) at least one antibody that bindsto IL-19, IL-20, IL-24, IL20Ra, IL-20Rb, or IL-10R2 (wherein any numberof the antibodies listed in 2) may be selected in any combination). Inanother aspect, a pharmaceutical formulation contains two or more activecompounds having complementary activities. For example, in oneembodiment, a pharmaceutical formulation may comprise 1) at least oneantagonist of IL-22, e.g., an antibody that binds to IL-22 and/or anantibody that binds to IL-22R; and 2) an antagonist of TNF-α or IL-12.In yet another aspect, a pharmaceutical formulation containing more thanone active compound may comprise a cytotoxic agent or growth inhibitoryagent.

F. Methods of Treatment

Therapeutic methods using any of the above compositions orpharmaceutical formulations are provided. Such methods include in vitro,ex vivo, and in vivo therapeutic methods, unless otherwise indicated. Invarious aspects, methods of stimulating or inhibiting an IL-23-mediatedsignaling pathway are provided. Methods of stimulating or inhibiting aTh_(IL-17) cell function are provided. Methods of treating inflammatoryand/or autoimmune disorders are also provided. Methods of treatingdisorders associated with IL-23 or IL-22 signaling are further provided.Methods of treating Th_(IL-17)-mediated disorders are also provided.These and other aspects of the invention are provided below.

In one aspect, a method of stimulating an IL-23 mediated signalingpathway in a biological system is provided, the method comprisingproviding an IL-22 agonist to the biological system. Biological systemsinclude, e.g., mammalian cells in an in vitro cell culture system or inan organism in vivo. Exemplary biological systems that model psoriasisare provided in the Examples and include reconstituted human epidermis(RHE) (Example 14) or animal models (Example 16). In one embodiment, anIL-22 agonist is IL-22. In another aspect, a method of inhibiting anIL-23-mediated signaling pathway in a biological system is provided, themethod comprising providing an IL-22 antagonist to the biologicalsystem. In one embodiment, the antagonist of IL-22 is an antibody, e.g.,a neutralizing anti-IL-22 antibody and/or a neutralizing anti-IL-22Rantibody.

In another aspect, a method of stimulating a Th_(IL-17) cell function isprovided, the method comprising exposing a Th_(IL-17) cell to an IL-22agonist. In one embodiment, an IL-22 agonist is IL-22. In anotheraspect, a method of inhibiting a Th_(IL-17) cell function is provided,the method comprising exposing a Th_(IL-17) cell to an IL-22 antagonist.In one embodiment, the IL-22 antagonist is an antibody, e.g., aneutralizing and-IL-22 antibody and/or a neutralizing anti-IL-22Rantibody. Exemplary Th_(IL-17) cell functions include, but are notlimited to, stimulation of cell-mediated immunity (delayed-typehypersensitivity); recruitment of innate immune cells, such as myeloidcells (e.g., monocytes and neutrophils) to sites of inflammation; andstimulation of inflammatory cell infiltration into tissues. In oneembodiment, a Th_(IL-17) cell function is mediated by IL-23.

In yet another aspect, a method of treating inflammation is provided,the method comprising administering to a mammal in need of suchtreatment an effective amount of a pharmaceutical formulation comprisingan antagonist of IL-22. In one embodiment, the antagonist of IL-22 is anantibody, e.g., a neutralizing anti-IL-22 antibody and/or a neutralizinganti-IL-22R antibody. Inflammation includes, but is not limited to,autoimmune inflammation (inflammation associated with an autoimmunedisorder), chronic inflammation, skin inflammation, arthriticinflammation (including inflammation associated with rheumatoidarthritis), and systemic inflammatory response. In one embodiment, theinflammation is mediated by IL-23.

In yet another aspect, a method of treating an autoimmune disorder isprovided, the method comprising administering to a mammal in need ofsuch treatment an effective amount of a pharmaceutical formulationcomprising an antagonist of IL-22. In one embodiment, the antagonist ofIL-22 is an antibody, e.g., a neutralizing anti-IL-22 antibody and/or aneutralizing anti-IL-22R antibody. Autoimmune disorders include, but arenot limited to, connective tissue disease, multiple sclerosis, systemiclupus erythematosus, inflammatory arthritis (e.g., rheumatoidarthritis), autoimmune pulmonary inflammation, Guillain-Barre syndrome,autoimmune thyroiditis, insulin-dependent diabetes mellitus, uveitis,myasthenia gravis, graft-versus-host disease, autoimmune inflammatoryeye disease, psoriasis, arthritis associated with autoimmunity (e.g.,rheumatoid arthritis), autoimmune inflammation of the brain, andinflammatory bowel disease. In one embodiment, the autoimmune disorderis an IL-23-mediated autoimmune disorder.

In a particular aspect, methods for the treatment of psoriasis and/ordisorders characterized by psoriatic symptoms are provided. Psoriasis isconsidered an autoimmune disease in which T-cells of the immune systemrecognize a protein in the skin and attack the area where that proteinis found, causing the too-rapid growth of new skin cells and painful,elevated, scaly lesions. These lesions are characterized byhyperproliferation of keratinocytes and the accumulation of activatedT-cells in the epidermis of the psoriatic lesions. Although the initialmolecular cause of disease is unknown, genetic linkages have been mappedto at least 7 psoriasis susceptibility loci (Psor1 on 6p21.3, Psor2 on17q, Psor3 on 4q, Psor4 on 1 cent-q21, Psor5 on 3q21, Psor6 on 19p13,and Psor7 on 1p). Some of these loci are associated with otherautoimmune/inflammatory diseases, including rheumatoid arthritis, atopicdermatitis, and inflammatory bowel disease (IBD). Current approaches tothe treatment of psoriasis include the administration of IL-12 or TNF-αantagonists. See, e.g., Nickoloff et al. (2004) J. Clin. Invest.113:1664-1675; Bowcock et al. (2005) Nat. Rev. Immunol. 5:699-711;Kauffman et al. (2004) J. Invest. Dermatol. 123:1037-1044. The dataprovided herein, however, implicate a distinct IL-23/IL-22 signalingpathway in the pathogenesis of psoriasis. Accordingly, therapeutics thatmodulate this signaling pathway may provide an alternative to or maycomplement other approaches to psoriasis treatment.

In one embodiment, a method of treating psoriasis comprisesadministering to a patient an effective amount of a pharmaceuticalformulation comprising an IL-22 antagonist. In one embodiment, theantagonist of IL-22 is an antibody, e.g., a neutralizing anti-IL-22antibody and/or a neutralizing anti-IL-22R antibody. In variousembodiments, the method further comprises administering (either in thesame pharmaceutical formulation or a separate pharmaceuticalformulation) at least one additional therapeutic agent. In one suchembodiment, the additional therapeutic agent is at least one antagonistof a cytokine selected from IL-19, IL-20, and IL-24. Such antagonistsinclude, but are not limited to, an antibody that binds IL-19, IL-20,IL-24, IL-20Ra, IL-20Rb, or IL-10R2. Any number of such antibodies maybe selected in any combination. In another embodiment, the additionaltherapeutic agent is an agent known to be effective in the treatment ofpsoriasis. Certain of such therapeutic agents are described, e.g., inNickoloff et al. (2004) J. Clin. Invest. 113:1664-1675; Bowcock et al.(2005) Nat. Rev. Immunol. 5:699-711; and Kauffman et al. (2004) J.Invest. Dermatol. 123:1037-1044. Such agents include, but are notlimited to, a therapeutic agent that targets T cells, e.g., efalizumaband/or alefacept; an antagonist of IL-12, e.g., a blocking antibody thatbinds IL-12 or its receptor, and an antagonist of TNF-α, e.g., ablocking antibody that binds TNF-α or its receptor.

In yet another aspect, a method of inhibiting tumor progression isprovided, the method comprising administering to a mammal an effectiveamount of a pharmaceutical formulation comprising an antagonist ofIL-22. In one embodiment, the antagonist of IL-22 is an antibody, e.g.,a neutralizing anti-IL-22 antibody and/or a neutralizing anti-IL-22Rantibody. In one embodiment, the tumor progression is IL-23 mediated.

Compositions of the present invention (e.g., polypeptides, antibodies,antagonists, agonists and pharmaceutical formulations comprising any ofthe foregoing), are administered to a mammal, preferably a human, inaccord with known methods, such as intravenous administration as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation (intranasal,intrapulmonary) routes. Intravenous or inhaled administration ofpolypeptides and antibodies is preferred.

In certain embodiments, administration of an anti-cancer agent may becombined with the administration of a composition of the instantinvention. For example, a patient to be treated with a composition ofthe invention may also receive an anti-cancer agent (chemotherapeuticagent) or radiation therapy. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992). The chemotherapeutic agent may precede or followadministration of the composition or may be given simultaneouslytherewith. Additionally, an anti-estrogen compound such as tamoxifen oran anti-progesterone such as onapristone (see, EP 616812) may be givenin dosages known for such molecules.

It may be desirable to also administer antibodies against other immunedisease associated- or tumor associated-antigens, such as antibodiesthat bind to CD20, CD11a, CD18, ErbB2, EGFR, ErbB3, ErbB4, or vascularendothelial factor (VEGF). Alternatively, or in addition, two or moreantibodies binding the same or two or more different antigens disclosedherein may be coadministered to the patient. In certain embodiments, itmay be beneficial to also administer one or more cytokines to a patient.In certain embodiments, a composition of the invention is coadministeredwith a growth inhibitory agent. For example, the growth inhibitory agentmay be administered before, after, or contemporaneously withadministration of the composition. Suitable dosages for the growthinhibitory agent are those presently used and may be lowered due to thecombined action (synergy) of the growth inhibitory agent and thecomposition.

For the treatment or reduction in the severity of an immune disease, theappropriate dosage of a composition of the invention will depend on thetype of disease to be treated, as defined above, the severity and courseof the disease, whether the agent is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the compound, and the discretion of the attendingphysician. The compound is suitably administered to the patient at onetime or over a series of treatments.

For example, depending on the type and severity of a disease, about 1μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of a polypeptide or antibody isan initial candidate dosage for administration to a patient, whether,for example, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

G. Diagnostic Methods and Methods of Detection

In one aspect, a method of diagnosing psoriasis in a mammal is provided,the method comprising detecting the level of expression of a geneencoding an IL-22 or IL-22R polypeptide in a test sample of tissue cellsobtained from the mammal, wherein a higher expression level in the testsample as compared to a control sample (e.g., a sample of known normaltissue cells of the same cell type) indicates the presence of psoriasisin the mammal from which the test sample was obtained. The detection maybe qualitative or quantitative. In one embodiment, the test samplecomprises blood or serum. In one embodiment, detecting the level ofexpression of a gene encoding an IL-22 or IL-22R polypeptide comprises(a) contacting an anti-IL-22 or anti-IL-22R antibody with a test sampleobtained from the mammal, and (b) detecting the formation of a complexbetween the antibody and an IL-22 or IL-22R polypeptide in the testsample. The antibody may be linked to a detectable label. Complexformation can be monitored, for example, by light microscopy, flowcytometry, fluorimetry, or other techniques known in the art. The testsample may be obtained from an individual suspected of having psoriasis.

In one embodiment, detecting the level of expression of a gene encodingan IL-22 or IL-22R polypeptide comprises detecting the level of mRNAtranscription from the gene. Levels of mRNA transcription may bedetected, either quantitatively or qualitatively, by various methodsknown to those skilled in the art. Levels of mRNA transcription may alsobe detected directly or indirectly by detecting levels of cDNA generatedfrom the mRNA. Exemplary methods for detecting levels of mRNAtranscription include, but are not limited to, real-time quantitativeRT-PCR and hybridization-based assays, including microarray-based assaysand filter-based assays such as Northern blots.

In another embodiment, the present invention concerns a diagnostic kitcontaining an anti-IL-22 or anti-IL-22R antibody in suitable packaging.The kit preferably contains instructions for using the antibody todetect an IL-22 or IL-22R polypeptide. In one aspect, the diagnostic kitis a diagnostic kit for psoriasis.

H. Assays

1. Cell-Based Assays and Animal Models

Cell-based assays and animal models for immune diseases are useful inpracticing certain embodiments of the invention. Certain cell-basedassays provided in the Examples below are useful, e.g., for testing theefficacy of IL-22 antagonists or agonists.

In vivo animal models are also useful in practicing certain embodimentsof the invention. Exemplary animal models are also described in theExamples below. The in vivo nature of such models makes them predictiveof responses in human patients. Animal models of immune related diseasesinclude both non-recombinant and recombinant (transgenic) animals.Non-recombinant animal models include, for example, rodent, e.g., murinemodels. Such models can be generated by introducing cells into syngeneicmice using standard techniques, e.g., subcutaneous injection, tail veininjection, spleen implantation, intraperitoneal implantation,implantation under the renal capsule, etc.

Graft-versus-host disease models provide a means of assessing T cellreactivity against MHC antigens and minor transplant antigens.Graft-versus-host disease occurs when immunocompetent cells aretransplanted into immunosuppressed or tolerant patients. The donor cellsrecognize and respond to host antigens. The response can vary from lifethreatening severe inflammation to mild cases of diarrhea and weightloss. A suitable procedure for assessing graft-versus-host disease isdescribed in detail in Current Protocols in Immunology, above, unit 4.3.

An animal model for skin allograft rejection is a means of testing theability of T cells to mediate in vivo tissue destruction and a measureof their role in transplant rejection. The most common and acceptedmodels use murine tail-skin grafts. Repeated experiments have shown thatskin allograft rejection is mediated by T cells, helper T cells andkiller-effector T cells, and not antibodies. Auchincloss, H. Jr. andSachs, D. H., Fundamental Immunology, 2nd ed., W. E. Paul ed., RavenPress, NY, 1989, 889-992. A suitable procedure is described in detail inCurrent Protocols in Immunology, above, unit 4.4. Other transplantrejection models which can be used to test the compounds of theinvention are the allogeneic heart transplant models described byTanabe, M. et al, Transplantation (1994) 58:23 and Tinubu, S. A. et al,J. Immunol. (1994) 4330-4338.

Contact hypersensitivity is a simple in vivo assay for cell mediatedimmune function (delayed type hypersensitivity). In this procedure,cutaneous exposure to exogenous haptens which gives rise to a delayedtype hypersensitivity reaction which is measured and quantitated.Contact sensitivity involves an initial sensitizing phase followed by anelicitation phase. The elicitation phase occurs when the T lymphocytesencounter an antigen to which they have had previous contact. Swellingand inflammation occur, making this an excellent model of human allergiccontact dermatitis. A suitable procedure is described in detail inCurrent Protocols in Immunology, Eds. J. E. Cologan, A. M. Kruisbeek, D.H. Margulies, E. M. Shevach and W. Strober, John Wiley & Sons, Inc.,1994, unit 4.2. See also Grabbe, S. and Schwarz, T, Immun. Today 19 (1):37-44 (1998).

Additionally, the compositions of the invention can be tested on animalmodels for psoriasis-like diseases. For example, compositions of theinvention can be tested in the scid/scid mouse model described by Schon,M. P. et al, Nat. Med. (1997) 3:183, in which the mice demonstratehistopathologic skin lesions resembling psoriasis. Another suitablemodel is the human skin/scid mouse chimera prepared as described byNickoloff, B. J. et al, Am. J. Path. (1995) 146:580. Another suitablemodel is described in Boyman et al., J Exp Med. (2004) 199(5):731-6, inwhich human prepsoriatic skin is grafted onto AGR129 mice, leading tothe development of psoriatic skin lesions.

Knock out animals can be constructed which have a defective or alteredgene encoding a polypeptide identified herein, as a result of homologousrecombination between the endogenous gene encoding the polypeptide and aDNA molecule in which that gene has been altered. For example, cDNAencoding a particular polypeptide can be used to clone genomic DNAencoding that polypeptide in accordance with established techniques. Aportion of the genomic DNA encoding a particular polypeptide can bedeleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

2. Screening Assays for Drug Candidates

Screening assays for drug candidates are designed to identify compoundsthat bind to or complex with a polypeptide identified herein or abiologically active fragment thereof, or otherwise interfere with theinteraction of a polypeptide with other cellular proteins. Suchscreening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates. Small molecules contemplatedinclude synthetic organic or inorganic compounds, including peptides,preferably soluble peptides, (poly)peptide-immunoglobulin fusions, and,in particular, antibodies including, without limitation, poly- andmonoclonal antibodies and antibody fragments, single-chain antibodies,anti-idiotypic antibodies, and chimeric or humanized versions of suchantibodies or fragments, as well as human antibodies and antibodyfragments. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart. All assays are common in that they call for contacting a testcompound with a polypeptide identified herein under conditions and for atime sufficient to allow the polypeptide to interact with the testcompound.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, a polypeptide or the test compound is immobilized on a solidphase, e.g., on a microliter plate, by covalent or non-covalentattachments. Non-covalent attachment generally is accomplished bycoating the solid surface with a solution of the polypeptide or testcompound and drying. Alternatively, an immobilized antibody, e.g., amonoclonal antibody specific for a polypeptide to be immobilized, can beused to anchor the polypeptide to a solid surface. The assay isperformed by adding the non-immobilized component, which may be labeledby a detectable label, to the immobilized component, e.g., the coatedsurface containing the anchored component. When the reaction iscomplete, the non-reacted components are removed, e.g., by washing, andcomplexes anchored on the solid surface are detected. When theoriginally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labelledantibody specifically binding the immobilized complex.

If the test compound interacts with but does not bind to a particularpolypeptide identified herein, its interaction with that protein can beassayed by methods well known for detecting protein-proteininteractions. Such assays include traditional approaches, such as,cross-linking, co-immunoprecipitation, and co-purification throughgradients or chromatographic columns. In addition, protein-proteininteractions can be monitored by using a yeast-based genetic systemdescribed by Fields and co-workers [Fields and Song, Nature (London)340, 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA 88,9578-9582 (1991)] as disclosed by Chevray and Nathans, Proc. Natl. Acad.Sci. USA 89, 5789-5793 (1991). Many transcriptional activators, such asyeast GAL4, consist of two physically discrete modular domains, oneacting as the DNA-binding domain, while the other one functioning as thetranscription activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

To identify compounds that interfere with the interaction of apolypeptide identified herein and other intra- or extracellularcomponent(s), a reaction mixture may be prepared containing thepolypeptide and the component under conditions allowing for theinteraction of the polypeptide with the component. To test the abilityof a test compound to inhibit the interaction, the reaction mixture isprepared in the absence and in the presence of the test compound. Ifthere is a decrease in the interaction of the polypeptide with thecomponent in the presence of the test compound, then the test compoundis said to inhibit the interaction of the polypeptide with thecomponent.

In certain embodiments, methods for identifying agonists or antagonistsof an IL-22 or IL-22R polypeptide comprise contacting an IL-22 or IL-22Rpolypeptide with a candidate agonist or antagonist molecule andmeasuring a detectable change in one or more biological activitiesnormally associated with the IL-22 or IL-22R polypeptide. Suchactivities include, but are not limited to, those described in theExamples below.

3. Antibody Binding Assays

Antibody binding studies may be carried out in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of target protein in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies preferably are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

Immunohistochemistry may also be used to determine the cellular locationof an antigen to which an antibody binds. For immunohistochemistry, thetissue sample may be fresh or frozen or may be embedded in paraffin andfixed with a preservative such as formalin, for example, Articles ofManufacture

In another aspect, an article of manufacture comprising compositionsuseful for the diagnosis or treatment of the disorders described aboveis provided. The article of manufacture comprises a container and aninstruction. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for diagnosing or treating the condition and may havea sterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agent in the composition is usually apolypeptide, an antibody, an agonist, or an antagonist of the invention.An instruction or label on, or associated with, the container indicatesthat the composition is used for diagnosing or treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

In one embodiment, the invention provides an article of manufacture,comprising:

(a) a composition of matter comprising an agonist or antagonist of IL-22or IL-22R;

(b) a container containing said composition; and

(c) a label affixed to said container, or a package insert included insaid container, referring to the use of said antagonist in the treatmentof an immune-related disease or cancer. The composition may comprise aneffective amount of the antagonist.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

III. Examples

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Generation of Anti-IL-22 and Anti-IL-22R Antibodies

This example illustrates preparation of monoclonal antibodies thatspecifically bind IL-22 or IL-22R. Techniques employed for producing themonoclonal antibodies were based on those known in the art and aredescribed, for instance, in Goding, supra. Immunogens employed were fulllength purified human IL-22 (hIL-22) or full length purified humanIL-22R (hIL-22R). Briefly, mice were immunized with about 1-100micrograms of the hIL-22 or hIL-22R immunogen emulsified in adjuvant.The immunized mice were then boosted 10 to 12 days later with additionalimmunogen emulsified in adjuvant. Serum samples were periodicallyobtained from the mice for testing in ELISA assays to detect anti-IL-22or IL-22R antibodies.

After a suitable antibody titer was detected, the animals “positive” forantibodies were sacrificed and the spleen cells harvested. The spleencells were then fused (using 35% polyethylene glycol) to a murinemyeloma cell line. The fusions generated hybridoma cells which werecloned and cultured in medium containing HAT (hypoxanthine, aminopterin,and thymidine). The hybridoma cells were screened in an ELISA forreactivity against IL-22 or IL-22R. (See FIG. 5.) A listing of theantibodies produced by those hybridomas and their respective propertiesis found in FIG. 5.

Example 2 IL-22 Signaling is Blocked by Anti-IL-22 Antibodies

STAT3 activation is a hallmark of IL-22 receptor activation andintracellular signaling. Antibodies generated against human IL-22 weretested for the ability to block IL-22-induced STAT3 activation. 293 Tcells expressing the human IL-22 receptor heterodimer (hIL-22R/hIL-10R2)were plated at 0.2×10̂6/well in a 24 well plate. Cells were transfectedwith a STAT3 Luciferase reporter (TK-SIE-SRE-S) using Lipofectamine2000™ (Invitrogen). Therefore, when STAT3 is activated, the cells willproduce luciferase, an enzymatic activity that can be detected by theaddition of luciferin. A reduction of luciferase activity means thatSTAT3 is blocked. The next day 0.5 nM of hIL-22 (R&D Systems) was addedto each well along with 20 μg/ml of antibody. Sixteen hours later thecells were lysed and samples read on a luminometer. Data shown in FIG. 6is luciferase activity relative to Renilla internal control, which is ameasure of relative STAT3 activation. As shown in FIG. 6, the antibodies3F11.3, 11H4.4, and 8E11.9 had significant blocking ability.

Example 3 Dose Versus Response of Anti-IL-22 Antibodies

A dose range of antibodies generated against human IL-22 were tested forthe ability to block human IL-22 in a STAT3 activation assay. 293 cellsexpressing hIL-22R/hIL-10R2 were plated at 0.2×10̂6/well in a 24 wellplate. Cells were transfected with a STAT3 Luciferase reporter(TK-SIE-SRE-S) using Lipofectamine 2000™ (Invitrogen). The next day 0.5nM of hIL-22 (R&D Systems) was added to each well along with varyingconcentrations of the anti-IL-22 antibodies 3F11, 8E11 or 11H4. Theconcentration range for the antibody began at 40 μg/ml with 2-folddilutions to a final concentration of 0.012 μg/ml. Sixteen hours laterthe cells were lysed and samples read on a luminometer. The threeantibodies show a similar dose/response curve for blocking STAT3activation, as shown in FIG. 7.

Example 4 Dose Versus Response of Anti-IL-22 Antibodies

A dose range of antibodies generated against human IL-22 were tested forthe ability to block murine IL-22 (mIL-22) in a STAT3 activation assay.293 cells expressing mIL-22R/mIL-10Rb were plated at 0.2×10̂6/well in a24 well plate. Cells were transfected with a STAT3 Luciferase reporter(TK-SIE-SRE-S) using Lipofectamine 2000™ (Invitrogen). The next day 0.5nM of mIL-22 (polyhistidine tagged) was added to each well along withvarying concentrations of 3F11, 8E11 or 11H4 antibody. The concentrationrange for the antibody started at 40 μg/ml with 2-fold dilutions to0.012 μg/ml. Sixteen hours later the cells were lysed and samples readon a luminometer. FIG. 8 shows that the anti-IL-22 antibodiescross-reacted with murine IL-22 and showed a similar, but not as robust,dose/response curve. This shows that the anti-IL-22 antibodies can beused in murine experiments.

Example 5 Affinity of Anti-IL-22 for Human IL-22

FIG. 9 shows the affinity of anti-IL-22 for human IL-22. The affinitywas measured by BIACore analysis. Various amounts of anti-IL-22 IgGswere immobilized on a CM 5 chip (845 RU (reference units) for 11H4 IgG,1933 RU for 8E11 IgG, & 7914 RU for 3F11 IgG) viaN-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) coupling chemistry. Two-fold serial dilutionsof IL-22 were prepared covering the range of 0.5-250 nM. The antigensamples were injected over the IgG-immobilized surface at a flow rate of20 μl/min for 6 minutes, and the bound complexes were allowed todissociate for 10 minutes. The IgG surfaces were regenerated with 10 mMGly, pH 1.5 after each round of antigen injection. As a negative controlflow cell, an irrelevant IgG (3A5 RF graft) was immobilized forbackground response subtraction. The running buffer, PBS containing0.05% Tween 20 with 0.01% NaN3 was used for all sample dilutions and thebinding experiment was done at 25° C. The data was analyzed by globalfitting with a 1:1 binding model. These results show that the anti-IL-22antibodies have very good affinity for human IL-22.

Example 6 Anti-IL-22 Antibodies Detect IL-22 in the Cell

Antibodies against IL-22 were tested for the ability to detectintracellular IL-22. For intracellular FACS staining of IL-22, thefollowing 293 cell lines were used: Cells expressing hIL-22-GFP,mIL-22-GFP, mIL20-GFP, and GFP only. The antibodies tested wereanti-human IL-22 antibodies 3F11, 8E11, and 17F6. Mouse anti-gp120 wasused as an isotype control. The secondary antibody used was anti-mouseIgG-PE from Jackson labs. Cells were incubated with Brefeldin A for 2hours, washed in PBS, and then fixed with 2% paraformaldehyde overnightat 4° C. Cells were then washed in PBS, and incubated in 5 ml 0.2%Tween-20 for 30 minutes at 37° C. Antibody staining was carried out for30 minutes at 4° C., then washed with Tween-20 solution. Cells wereresuspended in FACS buffer and analyzed on a FACScan. FIG. 10 shows theFACS results. The FACS results show that antibodies 3F11 and 8E11 causea shift in the cell staining pattern, indicating that these antibodiesbind both murine and human intracellular IL-22.

The anti-IL-22 antibody 3F11 was used in additional cell stainingexperiments. The 3F11 antibody was conjugated with Alexa 647, aphycoerythrin fluorophore. Mouse IgG2a conjugated to Alexa 647 was usedas an isotype control (Caltag). 293 cell lines expressing hIL-22-GFP andGFP only were assayed for 3F11 antibody binding. The 293 cells werefixed with 2% paraformaldehyde for 30 minutes, then washed twice withPBS/2% FCS. Cells were resuspended in 0.5% saponin for 15 minutes.Normal mouse serum was added for another 15 minutes, then antibodieswere added at 0.5 μg/million cells for 30 minutes. Cells were washed andresuspended in FACS buffer and analyzed on a FACScan. FIG. 11 shows inthe lower left panel a shift in cells into the upper right quadrant.This result indicates that the conjugated 3F11 antibody is binding tointracellular IL-22.

Example 7 Expression of IL-22 in Th1 T Cells

When CD4+ T cells mature from thymus and enter into the peripheral lymphsystem, they generally maintain their naïve phenotype beforeencountering antigens specific for their T cell receptor (TCR) [Sprentet al., Annu Rev Immunol. (2002); 20:551-79]. The binding of the TCR tospecific antigens presented by antigen-presenting cells (APC), causes Tcell activation. Depending on the environment and cytokine stimulation,CD4+ T cells can differentiate into a Th1 or Th2 phenotype and becomeeffector or memory cells [Sprent et al., Annu Rev Immunol. (2002);20:551-79 and Murphy et al., Nat Rev Immunol. (2002) December;2(12):933-44]. This process is known as primary activation. Havingundergone primary activation, CD4+ T cells become effector or memorycells, and they maintain their phenotype as Th1 or Th2. Once these cellsencounter antigen again, they undergo secondary activation, but thistime the response to antigen will be quicker than the primary activationand results in the production of effector cytokines as determined by theprimary activation [Sprent et al., Annu Rev Immunol. (2002); 20:551-79and Murphy et al., Annu Rev Immunol. 2000; 18:451-94]. Studies havefound during the primary and secondary activation of CD4+ T cells theexpression of certain genes is variable [Rogge et al., Nature Genetics.25, 96-101 (2000) and Ouyang et al., Proc Natl Acad Sci USA. (1999)March 30; 96(7):3888-93].

For primary activation conditions, naïve T cells may be activated by Ovaand APC. RNA isolated from cells in this condition can provideinformation about what genes are differentially regulated during theprimary activation, and what cytokines affect gene expression during Th1and Th2 development. After primary activation, the CD4+ T cells may bemaintained in culture. As the previous activation and cytokine treatmenthas been imprinted into these cells, they have become either effector ormemory cells. During this period, because there are no APCs or antigens,the CD4+ T cells enter a resting stage. This resting stage providesinformation about the differences between naïve vs. memory cells, andresting memory Th1 vs. resting memory Th2 cells. The resting memory Th1and Th2 cells then undergo secondary activation with anti-CD3/CD28antibodies or stimulation with IL12/IL18 cytokines. These conditionsprovide information about the differences between activated naïve andactivated memory T cells, and the differences between activated memoryTh1 vs. activated memory Th2 cells.

For the experiment shown in FIG. 12, splenocytes from DO11.10 mice wereisolated and activated by OVA in either Th1 conditions: [IL-12 (1ng/ml), IFN-γ, and IL-4 (1 μ/ml)]; Th0 conditions: [(anti-IL12,anti-IFN-γ, and anti-IL4)]; or Th2 conditions: [(anti-IL-12(0.5 μg/ml),anti-IFN-γ, and IL-4 (5 ng/ml]). RNA was harvested 48 hrs later (primarystimulation). The rest of the cells were maintained in the culture untilday 7, and then re-stimulated (secondary stimulation) by OVA andirradiated Balb/c splenocytes. A subset of the cells from Th1 conditionwere also stimulated by IL-12 and IL-18 alone. 48 hrs later RNA washarvested. The expression of IL-22, IFN-γ, and IL-4 in these RNA sampleswere analyzed by 5′ nuclease (TaqMan™) analysis. The expression wasfirst normalized against house keeping gene HPRT probes, then graphed asfold increase compared with the expression level from splenocytes. Theresult in shown in FIG. 12, and the data shows that IL-22 is highlyexpressed in Th1 cells upon secondary stimulation. Therefore anti-IL-22therapeutics would be useful in targeting these cells, either fortreatment of Th1 mediated disorders when it would be desirable to clearTh1 cells from the blood or as a diagnostic for Th1 mediated disorderswhen IL-22 is suspected to play a role.

Example 8 IL-22 is Produced by γδ T Cells

To analyze expression of IL-22 in γδ T cells, cells were isolated frommouse spleen and γδ T cells were separated by MACS sorting. GL4 is ananti-γδ TCR antibody which specifically activates γδ T cells(Becton-Dickenson) Qiagen MINI RNA isolation kit was used to isolate RNAfrom the cells for 5′ nuclease (TaqMan™) analysis. Master Mix one-stepRT-PCR Master Mix Reagent (Applied Biosystems; 4309169) was used and thehousekeeping genes RPL10 and SPF31 were used for normalization. Wholesplenocytes were used to determine the relative level of expression ofIL-22. FIG. 13 shows that IL-22 is highly expressed in γδ T cellsstimulated with GL4 antibody.

Example 9 IL-22 is Produced by Activated Human T Cells

Nucleic acid microarrays are useful for identifying differentiallyexpressed genes in diseased tissues as compared to their normalcounterparts. Using nucleic acid microarrays, test and control mRNAsamples from test and control tissue samples are reverse transcribed andlabeled to generate cDNA probes. The cDNA probes are then hybridized toan array of nucleic acids immobilized on a solid support. The array isconfigured such that the sequence and position of each member of thearray is known. For example, a selection of genes known to be expressedin certain disease states may be arrayed on a solid support.Hybridization of a labeled probe with a particular array memberindicates that the sample from which the probe was derived expressesthat gene. If the hybridization signal of a probe from a test (in thisinstance, activated CD4+ T cells) sample is greater than hybridizationsignal of a probe from a control (in this instance, non-stimulated CD4+T cells) sample, the gene or genes overexpressed in the test tissue areidentified. The implication of this result is that an overexpressedprotein in a test tissue is useful not only as a diagnostic marker forthe presence of the disease condition, but also as a therapeutic targetfor treatment of the disease condition.

The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. For example, the specificpreparation of nucleic acids for hybridization and probes, slides, andhybridization conditions are all detailed in PCT Patent ApplicationSerial No. PCT/US01/10482, filed on Mar. 30, 2001, and which is hereinincorporated by reference.

In this experiment, CD4+ T cells were purified from a single donor usingthe RossetteSep™ protocol from Stem Cell Technologies (Vancouver BC)which uses anti-CD8, anti-CD16, anti-CD19, anti-CD36 and anti-CD56antibodies used to isolate CD4+ T cells. Isolated CD4+ T cells wereactivated with an anti-CD3 antibody (used at a concentration that doesnot stimulate proliferation) together with either ICAM-1 or anti-CD28antibody. At 24 or 72 hours cells were harvested, RNA extracted andanalysis run on Affimax (Affymetrix Inc., Santa Clara, Calif.)microarray chips. Non-stimulated (resting) cells were harvestedimmediately after purification, and subjected to the same analysis.Genes were compared whose expression was upregulated at either of thetwo timepoints in activated vs. resting cells.

The results of this experiment are shown in FIG. 14. The microarrayresults support and compliment the data in Example 7. The Th1 T cellsproduce a large amount of IL-22 when stimulated, as opposed to the Th2cells which produce IL-4 or IL-5. This result would allow separation ofTh1 and Th2 related immune disorders based on the cytokine profile. Th1cells expressing IL-22 and IFN-γ could be treated by therapeuticsdirected to these cytokines, without affecting the Th2 cell population.

Example 10 Th1 Cells Express Intracellular IL-22

To determine the expression level of IL-22 in T cells by FACS,intracellular staining was carried out on murine Th1/Th2 cells. Primarysplenocytes were polarized to Th1 or Th2. For FACS staining, 1 millioncells were plated per well in a 96 well plate, and were treated withPMA/Ionomycin for 2 hours, then Brefeldin A for another 2 hours.Antibodies used were anti-human IL-22 (antibody 3F11.1) and anti-gp120as a control. Anti-mouse IFN-γ-FITC and anti-mouse IL-4-PE were obtainedfrom BD Bioscience (San Diego Calif.). PE-conjugated goat anti-mouse IgG(also from BD Bioscience) was used as a secondary antibody. Cells werefixed with 2% paraformaldehyde for 30 minutes, then washed twice withPBS/2% FCS. Cells were resuspended in 0.5% saponin for 15 minutes, thenantibodies were added at 0.5 ug/million cells for 30 minutes. Cells werethen washed twice and secondary antibody was added in 0.5% saponin for15 minutes. Finally, cells were washed and resuspended in FACS bufferand analyzed on a FACScan. FIG. 15 in the top panels show that Th1 cellscan be differentiated from Th2 cells. Th1 cells are positive for IFN-γ,negative for IL4, and positive for IL-22. Th2 cells are mostly negativefor IFN-γ, positive for IL4, and negative for IL-22.

Example 11 Generation of Anti-IL-22 Receptor (IL-22R)

To test binding of anti-IL-22R antibodies, 293 cells expressing hIL-22Rand cells expressing GFP were used. One million cells were stained withdifferent anti-hIL-22R antibodies at a concentration of 0.3 μg/millioncells. The antibodies tested were 7E9, 8A12, 8H11, and 12H5. Thesecondary antibody was Goat anti-Mouse PE conjugated (Jackson Labs) usedat a dilution factor of 1:200. Cells were washed and stained in FACSbuffer (0.5% BSA/PBS). Staining with the test antibodies was carried outfor 15 minutes at 4° C., then cells were washed, and secondary antibodywas added for another 15 minutes at 4° C. Cells were washed twice beforeanalysis on the FACScan. The results are shown in FIG. 16. For eachgraph in which the peaks do not overlap, the peak on the leftcorresponds to the control, and the peak on the right corresponds to thetest antibody. FIG. 16 shows that all of the four anti-IL-22R antibodiestested were positive for binding IL-22R on transfected 293 cells. Theantibodies 7E9, 8A12, 8H11, and 12H5 give good binding with very littlebackground.

Example 12 IL-22R Blocking Antibodies

To test for blocking activity of anti-IL-22R antibodies, a luciferasereporter construct (as described in Example 2) was used. If an antibodyhas blocking activity, STAT3 will not be activated and the luciferaseresponse will be low. Cells expressing hIL-22R/hIL10Rb were plated at0.2×10⁶/well in a 24-well plate and the luciferase reportersTK-SIE-SRE-S (0.8 μg/well) and RL-TK-Luc (0.16 μg/well) were transfectedinto cells. The following day, hIL-22 was added to the wells at 0.5 nM,and each antibody was added at a 20 μg/ml. The anti-IL-22R antibodiestested were; 7E9, 8A12, 8H11 and 12115. The control antibodies used wereGP120 and 11H4, an anti-hIL-22 antibody shown to have blocking activityin Example 2. Sixteen hours later the cells were lysed and samples readon a luminometer to detect the luciferase activity. FIG. 17 shows thatall four anti-IL-22R antibodies tested blocked the IL-22R-IL-22interaction.

Example 13 IL-22R is Expressed on Primary Keratinocytes

Keratinocytes are the cell population that overproliferates duringpsoriasis. Therapeutics that target keratinocytes are useful in thealleviation of psoriasis. Expression of IL-22R on primary humankeratinocytes was determined by FACS analysis. Normal human epidermalkeratinocytes (NHEK) donor lot 0526 were obtained from CascadeBiologics, passage #2, grown to 80% confluence, and were stained at300-600K cells per sample. Anti-IL-22R serum was used at a dilution of1:50 and pre-bleed serum was used at a dilution of 1:50 as the control.For IL10R2 staining, antibody from R&D (clone #90220, murine IgG1) wasused at 0.3 μg per sample with murine IgG1-PE isotype control (BDPharmingen #33815X). The secondary antibody for anti-IL-22R serum wasrat anti-mouse IgG1-PE (BD Pharmingen #550083), used at 0.1 ug persample. FIG. 18 shows that IL-22R and IL10R2 are expressed on NHEK.Therefore, blocking of the IL-22R or IL-22 may prove useful inalleviating disorders associated with keratinocyte hyperproliferation,such as psoriasis.

Example 14 Effect of IL-22 on Epidermal Cultures

Reconstituted human epidermis (RHE) can be used as a model for theeffects of cytokines on the skin. RHE and culture media were obtainedfrom MatTek Corporation (Ashland, Mass.). RHE was equilibrated overnight(20-22 hrs) with 0.9 ml media at 37° C., 5% CO₂, to recover fromshipping prior to start of the experiment and then cultured atair-liquid interface with 5 ml media at 37° C., 5% CO2. The effect ofIL-22 on RHE was assayed using three different conditions. IL-22 (1.2nM) or epidermal growth factor (EGF-R&D Systems) (1 nM) was added to themedia. The control consisted of untreated media. RHE was cultured for 4days, with a change of media every two days, adding fresh EGF or IL-22.RHE were harvested, fixed in 10% neutral buffered formalin (NBF)overnight, sectioned, and stained with hematoxylin and eosin (H&E). FIG.19 shows that IL-22 treatment causes thickening of the epidermis. Thisindicates that IL-22 causes hyperplasia, or proliferation of cells thatmake up the epidermis.

When these sections were stained for cytokeratin 16 (K16), a marker forkeratinocyte proliferation, the RHE treated with IL-22 showedsignificantly more staining for K16. K16 is expressed only inproliferating skin cells such as in psoriasis and wound healing(reviewed in Freedberg et al., Soc. Invest. Derm. 116:633-640 (2001)).FIG. 20 shows the K16 staining in IL-22 treated RHE relative tountreated and EGF treated RHE. The IL-22 treated RHE showed K16throughout the tissue, whereas the staining is localized in theuntreated and EGF treated sections.

Treatment of RHE with IL-22 also induces psoriasin, a gene highlyexpressed in psoriasis. Psoriasin (S100A7) was originally discovered asa protein expressed in psoriasis but not in normal skin (Madsen P., etal., J. Invest. Derm. 97: 701-712 (1991)). Psoriasin is expressed inactivated cultured and malignant keratinocytes, and in malignant breastepithelial cells (Watson et al., Int. J. of Biochem. and Cell Bio.30:567-571 (1998)). Current data support a role for psoriasininflammatory skin disease, chemotaxis, and breast tumor progression. Thecorrelation of psoriasin with psoriasiform hyperplasia of the skinsuggests a role in keratinocyte differentiation. Psoriasin may also bechemotactic, stimulating the neutrophil and CD4+ T-lymphocyteinfiltration of the epidermis that is a hallmark of psoriasis. FIG. 21shows that treatment of RHE with IL-22 induces high levels of psoriasinexpression. This result confirms the role that IL-22 and IL-22R play inpsoriasis.

The inducing effect of the IL-22 pathway on psoriasin can be blocked byantibodies directed to IL-22 or IL-22R. The anti-IL-22 antibody 8E11administered at a concentration of 20 μg/ml reduced psoriasin expressionto undetectable levels (see FIG. 23). When used at a concentration of 20μg/ml, the anti-IL-22R antibody (7E9) also significantly reducedpsoriasin expression as shown in FIG. 23.

The anti-IL-22 and anti-IL-22R antibodies were assayed to determine ifthey could reduce the epidermal thickening observed when RHE is treatedwith IL-22. The anti-IL-22 antibody (8E11) administered at aconcentration of 20 μg/ml showed significant reduction of epidermalthickening (see FIG. 24). RHE treated with IL-22 reached a thickness of80-90 μm, and treatment with anti-IL-22(8E11) antibody reduces the RHEthickness to 50-60 μm (FIG. 25). The anti-IL-22R antibody (7E9) alsoreduced skin thickening. When used at a concentration of 20 μg/ml,anti-IL-22R antibody reduced RHE thickness from 80-90 μm to 55-60 μm(FIG. 25). This data shows that anti-IL-22 or anti-IL-22R antibodies canalleviate symptoms associated with psoriasis, such as epidermalproliferation and thickening.

Example 15 Microarray Analysis of Genes Induced by IL-22

To determine what genes were induced by IL-22, normal human epidermalkeratinocytes (NHEK) derived from a single donor were plated and treatedat 70% confluence for 24 hrs with 20 ng/ml IL-22. Media and supplements(EpiLife®+HKGS) were obtained from Cascade Biologics™ (Portland, Oreg.).The cells were washed and lysed. Total RNA was purified from the NHEKcells using Qiagen RNeasy Mini Kit. The RNA was subjected to microarrayanalysis, and the amount of gene expression was quantified (See Example9 above for a description of microarray analysis).

Psoriasin is induced 81 fold upon stimulation by IL-22. SPR-2G isupregulated 11 fold. (See FIG. 22.) These results indicate that theIL-22 pathway is implicated in psoriasis. Therefore, antagonist andantagonist antibodies directed against IL-22 or IL-22R are useful inalleviating psoriasis.

Example 16 IL-23 Induces Hallmarks of Psoriasis in Vivo

A mouse model was used to compare the ability of IL-12 and IL-23 toinduce psoriatic skin features. C57Bl/6 mice were injectedsubcutaneously in the ear with 500 ng of either recombinant IL-12 orrecombinant IL-23 in a total volume of 20 μl PBS. Control mice wereinjected with 20 μl of PBS only. The mice were injected once every twodays for 16 days. Each experimental group consisted of five mice. Earthickness was measured before and at multiple time points afterinjection with a caliper (Mitutoyo America Corporation) and is reportedas mean±standard deviation. For this experiment and subsequentexperiments, statistical significance was calculated by one-way ortwo-way ANOVA using Prism software (GraphPad). All p values≦0.05 wereconsidered significant. Mouse ears were collected for routine histologicanalysis using hematoxylin-and-eosin (H&E) staining.

As shown in FIG. 26A, both IL-12 and IL-23 injection induced asignificant increase in ear thickness as early as one week following thefirst injection. For mice receiving IL-12, p was <0.001 (days 12, 14 and16 vs PBS control respectively). For mice receiving IL-23, p was <0.001(days 8, 12, 14 and 16 vs PBS control respectively). Histologic analysisrevealed that both IL-12 and IL-23 injected ears developed markedinflammatory cellular infiltration and epidermal thickening (acanthosis)compared to the PBS treated control group; however, there were someclear histologic differences between these two groups. First, IL-12induced mild to moderate acanthosis with a marked, predominantlymononuclear dermal inflammatory cellular infiltration (FIG. 26D, E)compared to the PBS control group (FIG. 26B, C), whereas IL-23 inducedmarked acanthosis with a mixed dermal inflammatory cellular infiltrationof many polymorphonuclear leukocytes (FIG. 26F, G), including bothneutrophils (arrows) and eosinophils. Epidermal hyperplasia and thepresence of polymorphonuclear leukocytes are histologic hallmarks ofpsoriasis in humans, as well as very common histologic findings in mousemodels of psoriasis. See P. C. van de Kerkhof et al., Dermatologica 174:224 (1987) and P. R. Mangan et al., Nature (2006) 441:235.

Example 17 IL-22 Acts Downstream of IL-23 in Vivo

To identify cytokines that potentially act downstream of IL-12 or IL-23,real-time PCR was used to examine the expression of a panel of cytokinesfrom ear skin samples injected with IL-12 or IL-23. Ear skin injectionsand histologic analysis were carried out as described in the precedingExample. On day 8 of the experiment, RNA was isolated from individualmouse ears and real-time PCR was performed to quantify the levels ofmRNA encoding IFN-γ, IL-17, and IL-22. Specifically, RNA was isolated byRNeasy Mini Kit (Qiagen, Valencia, Calif.) according to themanufacturer's instructions. Real-time RT-PCR was conducted using an ABI7500 Real-Time PCR System (Applied Biosystems, Foster City, Calif.) withprimers and probes using TaqMan™ One-Step RT-PCR Master Mix reagents(Applied Biosystems). Reactions were run in duplicate and samples werenormalized to the control housekeeping gene RPL-19 and reportedaccording to the ΔΔCt method.

As shown in FIG. 27A, IL-12 induced a significant increase in IFN-γexpression in the ear eight days after the first injection. IL-23induced IL-17 production and inhibited IFN-γ production relative to thePBS-treated control group (FIG. 27A). Interestingly, IL-22 was alsosignificantly up-regulated following IL-23 injection, but not afterinjection of IL-12 (FIG. 27A). These data suggested a link between IL-23and IL-22.

To confirm that the cytokines were produced by lymphocytes that hadinfiltrated the ear, lymphocytes were eluted out of the treated ears andcytokine production was measured by ELISA upon activation. Consistentwith the real-time RT-PCR data, cells from IL-23 injected earspreferentially produced IL-22 and IL-17, whereas cells from IL-12injected ears secreted large amount of IFN-γ (FIG. 28).

Example 18 IL-22 Induces Dermal Inflammation and Epidermal Hyperplasiain Vivo

To determine whether IL-22, like IL-23, is capable of inducing psoriaticskin features in vivo, mice were injected subcutaneously in the ear withIL-22 or with PBS alone, as described above in Example 16. As shown inFIG. 27B, IL-22 induced a significant increase in ear thickness comparedto the PBS treated group. IL-20, another cytokine from the IL-10 family,induced only a very mild and localized increase in ear thickness. Thisfinding was in contrast to a previous report where epidermal transgenicoverexpression of IL-20 induced marked epidermal hyperplasia, a resultthat suggested that IL-20 might potentially play a role in epidermalfunction as well as in psoriasis. See Blumberg et al., Cell 104:9(2001). Histologic analysis showed that IL-22 treated mouse ears had asimilar histologic appearance to ears in the IL-23 treated group shownin FIGS. 26F and G, exhibiting marked acanthosis and mixed dermalinflammatory cellular infiltration (FIG. 27G, H), including manyneutrophils (arrows) and some eosinophils. In contrast, IL-20 treatedears had only mild-moderate focal acanthosis with only moderate and veryfocal mixed inflammation (FIG. 27D, E) relative to the PBS treated group(FIG. 27C, F). These data suggested that IL-22 is essential forIL-23-induced skin inflammation and acanthosis.

Example 19 An Anti-IL-22 Blocking Antibody Significantly ReducedIL-23-Induced Acanthosis

To confirm that IL-23 acts through IL-22 to induce psoriatic skinfeatures, the effect of the anti-IL-22 monoclonal antibody 8E11 on IL-23induced dermal inflammation and acanthosis was examined. Mice wereinjected subcutaneously in the ear with IL-23 or PBS as described above(Example 16), except that the injections were carried out over a span of14 days. The mice were also injected intraperitoneally with 8E11 or withcontrol monoclonal antibody of the IgG1 isotype at a concentration of200 μg per mouse and at a frequency of once every two days for 14 days.On day 14, mouse ears were collected for histologic analysis using H&Estaining.

As shown in FIG. 29A, 8E11 (“anti-IL-22 mAb”) significantly reducedIL-23-induced epidermal acanthosis (*p<0.001) relative to treatment withcontrol IgG1 antibody. (Compare also FIGS. 29D and E (anti-IL-22 mAb)with B and C (control IgG1).) Furthermore, mice treated with anti-IL-22mAb also demonstrated a moderate decrease in dermal inflammation.However, mice treated with anti-IL-22 mAb still displayed a moderateinflammatory cellular infiltration when compared to ear skins treatedwith PBS. (Compare FIGS. 29D and E (anti-IL-22 mAb) with F and G (PBS).)

Example 20 IL-23-Induced Acanthosis was Significantly Reduced in IL-22Deficient Mice

To further confirm that IL-23 acts through IL-22 to induce psoriaticskin features, the effect of IL-23 on both wild type and IL-22 deficientmice was examined. IL-22 deficient mice (i.e., homozygous IL-22 knockoutmice, referred to as “IL-22^(−/−) mice”) were generated by targeted genedisruption according to the strategy depicted in FIG. 30A. Exons 1-4(closed boxes) of the IL-22 coding sequence were replaced with aneomycin resistance cassette flanked by loxP sites. Heterozygous micecarrying the conditional allele were crossed with a transgenic line inwhich the protamine 1 (Prm) promoter drove the Cre recombinase. Theconditional allele was excised during spermatogenesis in compoundheterozygous males (i.e., heterozygous for the conditional allele andthe PrmCre transgene). The compound heterozygous males were mated towild-type females, and the resulting progeny were screened for theexcised allele and the loss of the PrmCre transgene. Offspring werebackcrossed into C57Bl/6 background for at least six generations. Mousegenotypes were confirmed by PCR using the primers indicated in FIG. 30B.

IL-22 expression was examined at the mRNA and protein levels in Th cellsfrom wild type and IL-22^(−/−) mice. IL-22 mRNA expression was examinedin Th1, Th2, and Th_(IL-17) cells from wild type (“+/+”) and IL-22^(−/−)(“−/−”) mice (FIG. 30C) using RT-PCR, confirming that IL-22 mRNA was notdetected in IL-22^(−/−) mice. The expression of IL-22, IL-17, IFN-γ, andIL-4 was examined in Th1, Th2, and Th_(IL-17) cells from wild type(“WT”) and IL-22^(−/−) (“KO”) mice using ELISA. The results are shown inFIG. 30D for each of IL-22, IL-17, IFN-γ, and IL-4, as indicated at thetop of each graph, with filled bars and open bars indicating expressionlevels in WT and KO mice, respectively. Additionally, CD4 T cells fromIL-22^(−/−) mice were capable of being activated and differentiating toall T helper subsets and were able to produce normal levels of IL-17,IFN-γ, and IL-4 relative to wild type CD4 T cells. As expected, however,IL-22 was absent from IL-22^(−/−) CD4 T cells. IL-22^(−/−) mice wereobserved to develop normally and had similar lymphocyte composition anddevelopment in all major lymphoid organs examined as compared to wildtype mice. (Data not shown.)

IL-22^(−/−) mice and wild-type littermates were injected subcutaneouslyin the ear with IL-23 or PBS as described above (Example 16). On day 16,mouse ears were analyzed by routine histologic analysis. As shown inFIGS. 31A and B, IL-23 induced significantly less ear thickness andepidermal thickness in IL-22^(−/−) mice compared with the controlgroups. (IL-22^(−/−) mice are referred to in this figure and FIG. 32 as“KO” or “IL-22 KO”; wild type mice are referred to in this figure andFIG. 32 as “WT” or “IL-22 WT.”) By histological staining, both epidermalacanthosis and dermal inflammation were significantly reduced inIL-22^(−/−) mice (FIGS. 31E and F, respectively) compared toIL-23-treated wildtype littermates (FIGS. 31C and D, respectively). Incontrast to these results, IL-22 deficiency had no effect on IL-12induced ear skin inflammation at all. (FIG. 32.) Therefore, the datashow that IL-22 plays a crucial role in the dermal inflammation andepidermal acanthosis induced by IL-23, but not by IL-12.

Example 21 IL-23 Induces IL-22 Production from Various IL-23-ActivatedLymphocytes

To further investigate the ability of IL-23 to induce IL-22, variouslymphocyte populations were isolated and stimulated in vitro under theconditions indicated in FIG. 33. ELISA was performed to detect IL-22 inthe culture supernatants and is reported in FIG. 33A as mean±standarddeviation. The ability of IL-23 to induce IL-10 family cytokines otherthan IL-22 was also examined. Splenocytes from DO11.10 TCR transgenicmice were stimulated with 0.3 μM OVA peptide under indicated T-helpercell polarization conditions for 4 days, then rested for two days andrestimulated with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28(5 μg/ml) for another 2 days. Real-time RT-PCR was performed on RNAisolated from cells under the indicated conditions to quantify mouseIL-19, IL-20 and IL-24 mRNA expression. RNA from normal mousesplenocytes was also included as a control. As shown in FIG. 33B, IL-23did not induce expression of any other IL-10 family cytokines tested.

Example 22 IL-22 is a New Effector Cytokine from the Th_(IL-17) Lineage

Recently, IL-23 has been linked to the development of a new IL-17producing effector CD4⁺ T cell lineage (Th_(IL-17)). L. E. Harrington.,Nat. Immunol. 6:1123 (2005); H. Park, Nat. Immunol. 6:1133 (2005). IL-23is able to induce the Th_(IL-17) linage cells from naïve CD4+ T cells inthe presence of APC and antigen, but it is unable to initiate IL-17production when applied to purified naïve T cells activated withanti-CD3/anti-CD28. L. E. Harrington et al., Nat. Immunol. 6:1123(2005); M. Veldhoen et al., Immunity 24:179 (2006). Moreover, TGF-β andIL-6 have been suggested to be the de novo factors for Th_(IL-17) subsetdifferentiation. M. Veldhoen et al., Immunity 24:179 (2006).

Experiments were carried out to test whether IL-22 could be anadditional effector T cell cytokine induced by IL-23 under authentic TCRstimulation. CD4+ T cells from DO11.10 TCR transgenic mice wereactivated with 0.3 μM OVA peptide for four days under Th1-polarizing(IL-12 and anti-IL-4), Th2-polarizing (IL-4, anti-IL-12 and anti-IFN-γ),Th_(IL-17)-polarizing (IL-23, anti-IFN-γ and anti-IL-4) or Th0(anti-IL12/23 p40, anti-IFN-γ and anti-IL-4) conditions as previouslydescribed. L. E. Harrington et al., Nat Immunol 6:1123 (2005). RNA wasextracted from the cells and real-time PCR was performed to detectexpression of mRNA encoding various murine cytokines (indicated abovethe graphs in FIG. 34A). Additionally, ELISA was performed on theculture supernatants to detect expression of various cytokines at theprotein level. As shown in FIG. 34A, IL-17 was induced by IL-23, whereasIFN-γ and IL-4 were produced by Th1 and Th2 cells respectively. IL-22was produced, both at the mRNA and protein levels, from IL-17 producingTh_(IL-17) cells.

To determine whether IL-22 is a new effector cytokine from the fullycommitted Th_(IL-17) lineage, polarized T cells as described above wererested for two days and then restimulated for two days with plate-boundanti-CD3 (10 μg/ml) and soluble anti-CD28 (5 μg/ml) in the absence orpresence of IL-23. ELISA was performed to detect expression of themurine cytokines indicated above the graphs in FIG. 34B. The resultsdemonstrate that IL-17 was produced specifically from the Th_(IL-17)subset, even in the absence of IL-23, and IL-23 enhanced IL-17production. IL-23 failed to promote IL-17 production from committed Th1and Th2 cells. IL-22 demonstrated an identical expression pattern asIL-17, indicating IL-22 was a true effector cytokine expressed by thisnew Th_(IL-17) subset.

Previously, IL-23 receptor was reported to be expressed onactivated/memory T cells. C. Parham et al., J Immunol 168:5699 (2002).The above experiments did not exclude the possibility that IL-23 actedon memory T cells to produce IL-22. To address this more critically, theabove studies were repeated using naïve CD4⁺ T cells isolated fromDO11.10 TCR transgenic mice. Specifically, CD4⁺ T cells from Rag2^(−/−).DO11.10 TCR-transgenic mice were stimulated with OVA peptide-pulsedBALB/c splenic feeder cells (irradiated, T cells depleted) for 72 hoursin Th1-polarizing conditions (IL-12 and anti-IL-4), Th2-polarizingconditions (IL-4, anti-IL-12 and anti-IFN-γ), Th_(IL-17)-polarizing(IL-23, anti-IFN-γ and anti-IL-4), or other conditions as indicated inFIG. 35A. As shown in that figure, Th_(IL-17) cells produced the highestlevels of IL-22, while Th1 also secreted detectable levels of IL-22.Furthermore, addition of either IFN-γ or IL-4 completely abolished IL-17production; however, these two cytokines only moderately inhibited IL-22production (FIG. 35A). These data suggest potentially different pathwaysfor the induction of IL-17 versus IL-22 expression. However, fullyestablished Th_(IL-17) cells produced both IL-17 and IL-22 uponrestimulation for 48 hours in the indicated secondary conditions (FIG.35B). IL-23 further boosted the levels of these cytokines in a mannerthat could not be blocked by either IFN-γ or IL-4 (FIG. 35B). These dataconfirm the stability of this Th_(IL-17) lineage.

To further investigate whether IL-17 and IL-22 are produced by the samecells during activation, Th_(IL-17) cells were stimulated with PMA andionomycin, and antibodies to IL-22 or IL-17 were used for intracellularstaining. As shown in FIG. 35C, IL-17-producing cells were mainlyobserved from the Th_(IL-17) axis (left panel). IL-22-producing cellswere also preferentially detected from the Th_(IL-17) lineage (rightpanel). Costaining for both IL-22 and IL-17 revealed that a substantialportion of cells from the Th_(IL-17) lineage were producing both IL-22and IL-17 simultaneously, indicating that IL-22 and IL-17 are producedfrom the same cells.

As discussed above, recent studies also suggest that other factors fromAPC may be the primary driving force behind the differentiation ofIL-17-producing T cells from naïve CD4⁺ T cells, since IL-23 failed toinduce de novo IL-17 production from purified naïve CD4 T cells. M.Veldhoen et al., Immunity 24:179 (2006). Two of the factors critical forproduction of IL-17 from naïve CD4 T cells have been identified as TGF-βand IL-6. Id. To determine whether these factors were also critical forIL-22 production in mice, purified naïve CD4 T cells (>98%) werestimulated with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (5μg/ml). Consistent with published data, TGF-β and IL-6, rather thanIL-23, induced IL-17 production (FIG. 36A, right panel). Surprisingly,in contrast to the induction of IL-17, IL-22 was still only induced inthe presence of IL-23 and could not be induced by TGF-β and IL-6 (FIG.36A, left panel). These data suggest that transcription of IL-17 andIL-22 could be regulated differently. However, as previously reported,TGF-β and IL-6 could not establish a long term IL-17 producing T celllineage without IL-23 (FIG. 36B). The data thus demonstrate that IL-23might be one of the primary factors driving a T cell subset producingIL-22.

Next we examined whether a similar IL-22 producing T cell linage couldbe established from human CD4 T cells. We found that IL-23 could induceIL-22 secretion from purified naive human CD4⁺ T cells stimulated withanti-CD3/anti-CD28 under Th_(IL-17)-polarizing conditions (FIG. 36C,left panel). These cells could produce IL-22 upon restimulation withoutthe addition of exogenous IL-23 again (FIG. 36C, right panel),indicating the formation a stable T cell linage. Although these cellswere cultured under similar conditions as in the above murine studies,we could not detect IL-17 production above the assay limit (data notshown).

In conclusion, the data establish for the first time that IL-23 caninduce an IL-22-producing T cell subset from both murine and human naïveCD4 T cells. The production of IL-17 by this lineage depends upon otherenvironmental factors. While under authentic antigen and APC stimulatingconditions, IL-23 drove the T cell subset producing both IL-22 andIL-17. IL-23 also stimulated IL-22 production when naïve T cells wereactivated by anti-CD3 and ant-CD28. TGF-β and IL-6, which can inducetransient IL-17 production from naïve T cells but not long term lineagecommitment, failed to drive IL-22 production.

Example 23 IL-19, IL-20, and IL-24 Also Induce Epidermal Thickening

IL-22 belongs to a family of cytokines that include IL-19, IL-20, andIL-24, all of which show elevated expression in psoriatic skin. Thosecytokines were also tested to determine whether they, like IL-22, arecapable of inducing epidermal hyperplasia and acanthosis. RHE wascultured for four days and treated with IL-19, IL20, IL-22, or IL-24 at20 ng/ml or EGF at 6 ng/ml. The treated RHE was stained with H&E. Theresults are shown in FIG. 37A. All cytokines induced aconthosis of theviable nucleated epidermis, as denoted by the increased length of thedouble-headed arrows. Consistent with previous observations (above),IL-22 induced hypogranulosis, or a decrease in the granular cell layer(arrowheads), as well as hyalinization of the lower stratum corneum(asterisks). IL-22 also induced parakeratosis in RHE cultured for 7 days(data not shown). Hypogranulosis and parakeratosis are frequentlyobserved histological features of psoriasis. IL-19, IL-22, and IL-24induced only epidermal acanthosis with little or no apparent effect oneither the granular cell layer or the stratum corneum. EGF inducedepidermal aconthosis with hypergranulosis and compacting of thekeratinocytes within the stratum granulosum (arrows). Epidermalthickening induced by IL-19, IL20, IL-22, or IL-24 was quantified in anindependent experiment and is represented graphically in FIG. 38. IL-22had the greatest effect. The inflammatory cytokines TNF-α, IFN-γ, andIL-1β, which are thought to play a role in psoriasis, did not stimulatekeratinocyte proliferation in this RHE system (data not shown). Thus,those cytokines may play a secondary role in psoriasis or may play arole through a pathway independent of IL-19, IL-20, IL-22, and/or IL-24.

Immunohistochemistry was used to detect cytokeratin 16 (CK16), a markerof epidermal hyperplasia. IL-24, IL-22, and EGF induced CK16 expressionthroughout the non-cornified epidermis, while IL-19 and IL-20 onlyinduced CK16 expression in the basal zone. (FIG. 37B.)

Immunohistochemistry was also used to detect psoriasin (S100A7), one ofseveral S100 family proteins upregulated in certain hyperproliferativeand inflammatory skin conditions, including psoriasis. IL-19, IL-20,IL-22, and IL-24 all induced S100A7 expression in the suprabasalepidermis, with IL-22 and IL-24 having the greatest effect. (FIG. 37C).S100A7 staining was observed in the nuclei and cytoplasm of thekeratinocytes, with some protein also appearing to be extracellular. Theresults shown in FIGS. 37B and C were quantified and are displayedgraphically in FIGS. 37E and F.

Immunohistochemistry was also used to detect pY(705)-STAT3, thetransactivating form of STAT3. Activated STAT3 has been shown to beelevated in psoriatic lesional skin. IL-19, IL-20, IL-22, and IL-24 allinduced persistent STAT3 activation in RHE keratinocytes found in allviable cell layers, demonstrated by its nuclear localization. (FIG.37D).

Example 24 Blocking Antibodies to Receptors for IL-20 and IL-22 ReducePsoriasin Expression

Both IL-19 and IL-20 signal through a receptor heterodimer of IL20Ra andIL20Rb. IL20 also signals through a receptor heterodimer of IL-22R andIL-20Rb. IL-22 signals through a heterodimer of IL-22R and IL10R2. Cellsurface expression of these receptor components on keratinocytesisolated from RHE or from primary cultures of normal human epidermalkeratinocytes (NHEK, from donated neonatal foreskin) was examined byflow cytometry. The following monoclonal antibodies were used for flowcytometry: anti-IL20Ra (generated in mice for purposes of this study);anti-IL20Rb (generated in mice for purposes of this study); anti-IL-22Rantibody 7E9 (described above); and anti-IL-10R2 FAB874P (PE-conjugated)(R&D Systems, Minneapolis, Minn.). The results are shown in FIG. 39. Thereceptor component to which each antibody binds is shown in the upperright of each graph (IL-22R is designated as “IL-22R1”). IL-20Rb andIL10R2 were consistently expressed on the surface of NHEKs, regardlessof confluence, passage number or calcium levels in the medium. (FIG.39A.) In contrast, cell surface expression of both IL-20Ra and IL-22R1on NHEK varied from donor to donor, and was consistently at a relativelylow but detectable level. (FIG. 39A and data not shown.) Compared toexpression levels in monolayer NHEK, IL-20Ra and IL-22R were expressedat much higher levels on keratinocytes isolated from RHE (FIG. 39B). Thereasons for this difference are unknown. However, it is nonethelessclear that all of the receptor components analyzed are expressed onhuman keratinocytes. Expression of these receptor components on immunecells (T cells, B cells, natural killer cells, and monocytes) was notdetected. (Data not shown.) Thus, the ligands for these receptorcomponents likely provide a link between the immune system andkeratinocyte abnormalities.

To examine whether the above antibodies could block the effects ofIL-19, IL-20, and IL-22 treatment, as described in the precedingExample, 20 micrograms/ml of anti-IL20Ra, anti-IL20Rb, or anti-IL-22Rwas added to RHE culture media one hour prior to addition of 20 ng/ml ofIL-19, IL-20, or IL-22. RHE was then cultured for four days, with mediachanged at day two (4.5 ml fresh media including cytokine and antibody).The RHE was then stained by immunohistochemistry for psoriasin (S100A7).The results are shown in FIG. 40. IL-19, IL-20, and IL-22-treated RHEare shown in the first, second, and third rows, respectively. RHEpre-treated with anti-IL20Ra (αIL-20Ra), anti-IL20Rb (αIL-20Rb), oranti-IL-22R (αIL-22R1) are shown in the third, fourth, and fifthcolumns, respectively. No antibody controls and isotype controlantibodies are shown in the first and second columns.

The results show that either anti-IL20Ra or anti-IL20Rb effectivelyblocked IL-19-induced expression of psoriasin. Similarly, anti-IL-22Reffectively blocked IL-22-induced expression of psoriasin. Anti-IL20Rbeffectively blocked IL-20-induced expression of psoriasin, butanti-IL20Ra did not. Similarly, anti-IL-22R was unable to blockIL-20-induced expression of psoriasin.

To further investigate the effects of anti-IL-22R and anti-IL20Ra onIL-20-induced expression of psoriasin, RHE was pretreated with thoseantibodies either singly or in combination prior to treatment withIL-20. The results are shown in FIG. 41. As described above, eitheranti-IL-22R or anti-IL20Ra alone was unable to block IL-20-inducedexpression of psoriasin (second column, both panels). However, thecombination of both anti-IL20Ra and anti-IL-22R effectively blockedIL-20 induced expression of psoriasin, suggesting that IL-20Ra andIL-22R have complementary roles in IL-20 signaling in humankeratinocytes (lower left panel).

Example 25 IL-19, IL-20, IL-22, and IL-24 Induce Similar Gene ExpressionProfiles

To identify genes induced by IL-19, IL-20, IL-22, and IL-24, RHE wastreated with 20 ng/ml of IL-19, IL-20, IL-22, or IL-24 for four days.RNA was prepared, and cDNA was hybridized to Affymetrix U133 Plus GeneChips (Affymetrix, Santa Clara, Calif.), which contain 54,675 probesets.The data were analyzed for genes whose expression was increased by atleast 2-fold. IL-20, IL-22, and IL-24 showed similar gene expressionprofiles. Of the top 20 genes commonly induced by IL-20, IL-22, andIL-24, seven were genes previously reported to be associated withpsoriasis. Those genes are psoriasin (S100A7), S100A12, SCCA2, SERPINB4,CCL20, CD36, and Stat3.

To examine whether genes induced by IL-20, IL-22, and IL-24 showupregulation in psoriasis, the microarray analyses described above werecompared with a previous microarray study of psoriatic skin (Zhou et al.(2003) Physiol. Genomics 13:69-78). Because that study was performedusing a different microarray chip, only refseqs in common between thatstudy and the present study were compared. Out of 468 refseqs that wereupregulated in psoriatic skin, 356 were induced by IL-20, IL-22, andIL-24, and 188 of them were significant (p<0.05). Taken together, theabove studies demonstrate substantial overlap between genes that areinduced by IL-20, IL-22, and IL-24 and genes that are upregulated inpsoriatic skin.

Example 26 Deposit of Materials

The following hybridoma cell line has been deposited with the AmericanType Culture Collection, 10801 University Blvd., Manassas, Va.20110-2209 USA (ATCC):

Hybridoma/Antibody Designation ATCC No. Deposit Date Anti-IL-22 (3F11.3)PTA-7312 Jan. 13, 2006 Anti-IL-22 (11H4.4) PTA-7315 Jan. 13, 2006Anti-IL-22 (8E11.9) PTA-7319 Jan. 13, 2006 Anti-IL-22R (7E9.10.8)PTA-7313 Jan. 13, 2006 Anti-IL-22R (8A12.32) PTA-7318 Jan. 13, 2006Anti-IL-22R (8H11.32.28) PTA-7317 Jan. 13, 2006

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit. The cell line will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc. and ATCC, which assures (a) that access to the culturewill be available during pendency of the patent application to onedetermined by the Commissioner to be entitled thereto under 37 CFR §1.14and 35 USC §122, and (b) that all restrictions on the availability tothe public of the culture so deposited will be irrevocably removed uponthe granting of the patent.

The assignee of the present application has agreed that if the cultureon deposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the deposited cellline is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the material deposited, sincethe deposited embodiment is intended as a single illustration of certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims

1-2. (canceled)
 3. A method of treating an autoimmune disorder, whereinthe autoimmune disorder is not arthritis, the method comprisingadministering to a mammal an effective amount of a pharmaceuticalformulation comprising an antagonist of IL-22.
 4. The method of claim 3,wherein the IL-22 antagonist is an antibody that specifically bindsIL-22.
 5. The method of claim 4, wherein the antibody that specificallybinds IL-22 is (a) an antibody produced by a hybridoma selected from3F11.3 ATCC Accession No. PTA-7312), hybridoma 11H4.4 (ATCC AccessionNo. PTA-7315), and hybridoma 8E11.9 (ATCC Accession No. PTA-7319); (b)an affinity matured form of the antibody of (a); (c) an antigen-bindingfragment of the antibody of (a) or (b); or (d) a humanized form of theantibody of (a), (b), or (c).
 6. The method of claim 3, wherein theIL-22 antagonist is an antibody that specifically binds IL-22R.
 7. Themethod of claim 6, wherein the antibody that specifically binds IL-22Ris (a) an antibody produced by a hybridoma selected from 7E9 (ATCCAccession No. PTA-7313), hybridoma 8A12 (ATCC Accession No. PTA-7318),and hybridoma 8H11 (ATCC Accession No. PTA-7317); (b) an affinitymatured form of the antibody of (a); (c) an antigen-binding fragment ofthe antibody of (a) or (b); or (d) a humanized form of the antibody of(a), (b), or (c).
 8. The method of claim 3, wherein the IL-22 antagonistis IL-22BP.
 9. The method of claim 3, wherein the autoimmune disorder isinflammatory bowel disease.
 10. The method of claim 3, wherein theautoimmune disorder is psoriasis.
 11. The method of claim 10, whereinthe IL-22 antagonist is an antibody that specifically binds IL-22. 12.(Original The method of claim 11, further comprising administering atleast one antibody selected from an antibody that specifically bindsIL20Ra, an antibody that specifically binds IL20Rb, and an antibody thatspecifically binds IL-22R.
 13. The method of claim 10, wherein the IL-22antagonist is an antibody that specifically binds IL-22R.
 14. The methodof claim 13, further comprising administering at least one antibodyselected from an antibody that specifically binds IL-22, an antibodythat specifically binds IL20Ra, and an antibody that specifically bindsIL20Rb.
 15. A method of treating inflammation, wherein the inflammationis not arthritic inflammation, the method comprising administering to amammal an effective amount of a pharmaceutical formulation comprising anantagonist of IL-22.
 16. The method of claim 15, wherein the IL-22antagonist is an antibody that specifically binds IL-22.
 17. The methodof claim 16, wherein the antibody that specifically binds IL-22 is (a)an antibody produced by a hybridoma selected from 3F11.3 (ATCC AccessionNo. PTA-7312), hybridoma 11H4.4 (ATCC Accession No. PTA-7315), andhybridoma 8E11.9 (ATCC Accession No. PTA-7319); (b) an affinity maturedform of the antibody of (a); (c) an antigen-binding fragment of theantibody of (a) or (b); or (d) a humanized form of the antibody of (a),(b), or (c).
 18. The method of claim 15, wherein the IL-22 antagonist isan antibody that specifically binds IL-22R.
 19. The method of claim 18,wherein the antibody that specifically binds IL-22R is (a) an antibodyproduced by a hybridoma selected from 7E9 (ATCC Accession No. PTA-7313),hybridoma 8A12 (ATCC Accession No. PTA-7318), and hybridoma 8H11 (ATCCAccession No. PTA-7317); (b) an affinity matured form of the antibody of(a); (c) an antigen-binding fragment of the antibody of (a) or (b); or(d) a humanized form of the antibody of (a), (b), or (c).
 20. The methodof claim 15, wherein the IL-22 antagonist is IL-22BP.
 21. The method ofclaim 15, wherein the inflammation is autoimmune inflammation.
 22. Themethod of claim 15, wherein the inflammation is skin inflammation. 23.The method of claim 15, wherein the inflammation is chronicinflammation.
 24. A method of inhibiting tumor progression, the methodcomprising administering to a mammal an effective amount of apharmaceutical formulation comprising an antagonist of IL-22.
 25. Themethod of claim 24, wherein the IL-22 antagonist is an antibody thatspecifically binds IL-22.
 26. The method of claim 25, wherein theantibody that specifically binds IL-22 is (a) an antibody produced by ahybridoma selected from 3F11.3 (ATCC Accession No. PTA-7312), hybridoma11H4.4 (ATCC Accession No. PTA-7315), and hybridoma 8E11.9 (ATCCAccession No. PTA-7319); (b) an affinity matured form of the antibody of(a); (c) an antigen-binding fragment of the antibody of (a) or (b); or(d) a humanized form of the antibody of (a), (b), or (c).
 27. The methodof claim 25, wherein the IL-22 antagonist is an antibody thatspecifically binds IL-22R.
 28. The method of claim 27, wherein theantibody that specifically binds IL-22R is (a) an antibody produced by ahybridoma selected from 7E9 (ATCC Accession No. PTA-7313), hybridoma8A12 (ATCC Accession No. PTA-7318), and hybridoma 8H11 (ATCC AccessionNo. PTA-7317); (b) an affinity matured form of the antibody of (a); (c)an antigen-binding fragment of the antibody of (a) or (b); or (d) ahumanized form of the antibody of (a), (b), or (c).
 29. The method ofclaim 24, wherein the IL-22 antagonist is IL-22BP.
 30. A method ofstimulating an IL-23-mediated signaling pathway in a biological system,the method comprising providing an IL-22 agonist to the biologicalsystem.
 31. The method of claim 30, wherein the IL-22 agonist is IL-22.32. A method of inhibiting an IL-23-mediated signaling pathway in abiological system, the method comprising providing an IL-22 antagonistto the biological system.
 33. The method of claim 32, wherein the IL-22antagonist is an antibody that specifically binds IL-22.
 34. The methodof claim 32, wherein the IL-22 antagonist is an antibody thatspecifically binds IL-22R.
 35. A method of stimulating a Th_(IL-17) cellfunction, the method comprising exposing a Th_(IL-17) cell to an IL-22agonist.
 36. The method of claim 35, wherein the IL-22 agonist is IL-22.37. A method of inhibiting a Th_(IL-17) cell function, the methodcomprising exposing a Th_(IL-17) cell to an IL-22 antagonist.
 38. Themethod of claim 37, wherein the IL-22 antagonist is an antibody thatspecifically binds IL-22.
 39. The method of claim 37, wherein the IL-22antagonist is an antibody that specifically binds IL-22R.